E. coli plasmid DNA production

ABSTRACT

General methods and strains of bacteria are described that dramatically simplify and streamline plasmid DNA production. In one preferred embodiment, endolysin mediated plasmid extraction combined with flocculation mediated removal of cell debris and host nucleic acids achieves increased yield and purity with simplified downstream purification and reduced waste streams, thus reducing production costs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 12/601,504filed Nov. 23, 2009, now U.S. Pat. No. 9,017,966 issued Apr. 28, 2015,which is the U.S. national phase of PCT Appln. No. PCT/US2008/006553filed May 22, 2008, which claims the benefit of U.S. provisionalapplication Ser. No. 60/931,465 filed May 23, 2007, the disclosures ofwhich are hereby incorporated in their entirety by reference herein.

This application claims benefit of Provisional Patent Application Ser.No. 60/931,465 filed 23 May 2007

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with government support under Grant No.2 R44 GM072141-02, awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

SEQUENCE LISTING

The text file titled Autolysis_ST25.txt, created Nov. 23, 2009, and ofsize 10 KB, filed herewith, is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the production of covalently closedcircular (ccc) recombinant DNA molecules such as plasmids, cosmids,bacterial artificial chromosomes (BACs), bacteriophages, viral vectorsand hybrids thereof, and more particularly is methods for creating seedstocks, fermentation, cell lysis, and purification of plasmid DNA.

BACKGROUND OF THE INVENTION

The present invention relates to the production of covalently closedcircular (ccc) recombinant DNA molecules. Such molecules are useful inbiotechnology, transgenic organisms, gene therapy, therapeuticvaccination, agriculture and DNA vaccines.

With the invention in mind, a search of the prior art was conducted. E.coli plasmids have long been the single most important source ofrecombinant DNA molecules used by researchers and by industry. Today,plasmid DNA is becoming increasingly important as the next generation ofbiotechnology products (gene medicines and DNA vaccines) make their wayinto clinical trials, and eventually into the pharmaceuticalmarketplace. Plasmid DNA vaccines may find application as preventivevaccines for viral, bacterial, or parasitic diseases; immunizing agentsfor the preparation of hyper immune globulin products; therapeuticvaccines for infectious diseases; or as cancer vaccines.

Fermentation

Vector Backbones

Therapeutic plasmids typically contain a pMBI, ColE1 or pBR322 derivedreplication origin. Common high copy number derivatives have mutationsaffecting copy number regulation, such as rop (Repressor of primer gene)deletion, with a second site mutation that increases copy number (e.g.pMB1 pU CG to A point mutation, or ColE1 pMMI). Higher temperature (42°C.) can be employed to induce selective plasmid amplification with pUCand pMMI replication origins.

Nature Technology Corporation Fed-Batch Process:

Carnes, A E., Williams, J A 2006 World Patent Application WO2006023546discloses methods for fed-batch fermentation, in whichplasmid-containing E. coli cells are grown at a reduced temperatureduring part of the fed-batch phase, during which growth rate isrestricted, followed by a temperature up-shift and continued growth atelevated temperature in order to accumulate plasmid; the temperatureshift at restricted growth rate improves yield and purity of plasmid.

This process takes advantage of the temperature sensitivity of high copynumber plasmids. In the preferred process, the initial temperaturesetpoint is 30° C., at which the plasmid is maintained stably at lowlevels while biomass can accumulate efficiently. During this period, thespecific growth rate is controlled at approximately μ=0.12 h⁻¹ by anexponential feeding strategy. Induction of plasmid accumulation isperformed when the cell density is in the range of 25-60 OD₆₀₀ byshifting the temperature to 42° C. and continued exponential nutrientfeeding for up to 15 hours.

Plasmid yields prior to the temperature shift remain low. The specificplasmid yields after temperature shift are very high. Interestingly,after the temperature shift, the cells are able to toleratesignificantly higher quantities of plasmid than cells grown at aconstant temperature of 37° C. with the same media and feeding strategy.

The examples in the patent report yields up to 1.1 g/L achieved when thedisclosed process was used with a temperature shift from 30° C. to 42°C. The preferred process of Carnes and Williams, Supra, 2006 is alsodescribed in Carnes, A E, Hodgson C P, Williams J A. 2006 BiotechnolAppl Biochem 45:155-66 where volumetric yields of 1.5-2.1 g/L, andspecific yields as high as 43 mg plasmid/g dry cell weight (DCW) arereported. The plasmid DNA produced with the process is of a highquality, being 96% supercoiled or greater with no detectable deletion orother rearrangement. The method is simple, can be used with multiple pUCbased backbones, and does not require prescreening of individualcolonies for high producing cell lines. A key advantage of the induciblefed-batch process is that amplification of plasmid copy number aftersuitable biomass accumulation helps preserve quality and stabilize toxicplasmids, while maximizing yield. This is because selection pressure atthe cellular level is reduced during the biomass accumulation phase byminimizing the growth rate difference between monomer containing cellsand dimer, or rearranged plasmid or plasmid-free cells.

Fermentation Summary

High specific yields are very desirable since increased plasmid yieldper gram of bacteria, or increased plasmid relative to genomic DNA(utilizing the Carnes and Williams, Supra, 2006 process, up to 75% ofthe total DNA in the cell at harvest is plasmid DNA) lead directly tohigher final product purities. Further improvements in yield orincreases in the percentage plasmid per total DNA would further decreaseproduction cost, improve purity and simplify removal of genomic DNA(gDNA). Other fermentation processes for plasmid production are reviewedin Carnes et al., Supra, 2006, and Carnes A. E. BioProcess Intl 2005;3:36-44, and are included herein by reference.

Cell Disruption—Plasmid Release

The E. coli biomass generated by a fermentation process must be lysed torelease the plasmid DNA (pDNA). Cell disruption methods fall into twomain categories:

Physico-mechanical Chemical liquid shear detergents solid shear osmoticshock agitation with abrasives alkali treatment freeze-thawing enzymetreatment ultrasonication heat

The cell disruption method for plasmid isolation must be chosen suchthat minimal damage is inflicted on the pDNA product, and in most cases,it is also desired to avoid shearing of the host cell gDNA into smallerfragments that are more difficult to separate from pDNA. Thus, themethods available for plasmid purification are more limited compared tothe harsher methods that are often used for purifying smaller moleculessuch as proteins. Ideally the method releases a high yield of intactplasmid, while limiting release of difficult to remove impurities suchas gDNA.

Most of the above methods have been applied, either alone or combined,to pDNA purification. To date, the two most commonly used methods forpDNA recovery are alkaline lysis and heat lysis; additionally,detergents and enzyme (i.e. lysozyme) treatment are often used to aidheat lysis and other methods.

Alkaline Lysis

The standard alkaline lysis method of Birnboim H C, Doly J. 1979 NucleicAcids Res. 7:1513-23 is well known and is widely used withoutrestriction in molecular biology laboratories. Generally, a lysis timeof five minutes has been used with the standard alkaline lysis method;longer times have been known to cause irreversible denaturation of pDNA.A study (Ciccolini L A, Shamlou P A, Titchener-Hooker N J, Ward J M,Dunnill P. 1998 Biotechol Bioeng. 60: 768-770) on the time course ofstandard alkaline lysis was performed by measuring viscosity of a lysismixture as a function of time and by performing cell counts over a rangeof lysis times. The results indicate that for E. coli DH5α, completecell lysis occurs after about 40 sec and complete denaturation of gDNAtakes 80-120 sec after mixing with the lysis buffer. Longer reactiontimes were reported to lead to shear degradation of gDNA.

Thatcher D R, Hitchcock A, Hanak J A J, Varley D. 2003 U.S. Pat. No.6,503,738 describe a method to determine the optimum lysis pH value,which is about 0.2 pH below the “irreversible alkaline denaturationvalue”, defined as “the pH value at which no more than about 50% of thealkaline denatured pDNA fails to renature as determined by standardagarose gel electrophoresis”. The optimum lysis value can be differentfor various plasmid/host strain combinations.

The patent landscape includes various methods and devices aimed atperforming alkaline lysis at large scale. Insufficient mixing willresult in local pH extremes, causing irreversibly denatured plasmid.Mixing that is too aggressive can damage the pDNA and fragment gDNA. Atthe laboratory scale, mixing is performed gently by hand. Hand mixing atlarger scales is not possible because of the large volumes and lack ofreproducibility from person to person. Thus, batch mixing in amechanically agitated vessel is often used, but the viscous,non-Newtonian properties of the lysis mixture require someconsideration. Nienow A W, Hitchcock A G, Riley G L. 2003 U.S. Pat. No.6,395,516 discloses a specialized vessel design for mixing cell lysatethat utilizes baffles, low power number impellers, feed lines, andmonitoring the degree of lysis by measuring viscosity.

Continuous flow through devices have been employed as alternatives tothe challenge of achieving complete, but gentle mixing of large lysisvolumes in stirred tanks, and are perhaps more easily implemented infacilities that do not already contain specialized batch mixingequipment. Additionally, the lysis reaction time can be closelycontrolled by the residence time of tubing or pipe (e.g. as in Wan N C,McNeilly D S, Christopher C W. 1998 U.S. Pat. No. 5,837,529; Chevalier,M 2003 U.S. Pat. No. 6,664,049; Detraz N J F, Rigaut G. 2006 WorldPatent Application WO2006060282), or of the holding vessel (e.g. as inBrooks R C. 2004 U.S. Pat. No. 6,699,706) before the neutralizationstep.

Inline static mixers (motionless mixers) have long been used in industryand more recently have been applied for cell lysis. A static mixer is acylindrical tube containing stationary mixing elements. The mixingelements are shaped and positioned to combine materials as they flowthrough the mixer. Wan et al, Supra 1998 describes the use of staticmixers to achieve gentle mixing of a cell suspension with a lysissolution. Mixing of the cell suspension stream with the lysis bufferstream is completed rapidly and the degree of mixing and lysis time canbe adjusted by the number of mixing elements, flowrate, and length.Neutralization may occur in a second static mixer.

Chevalier, Supra, 2003 claims mixing methods that use only tubing,without the static mixers; instead, smaller diameter tubing is used andflowrates are adjusted to cause homogeneous mixing for the desiredcontact time.

Detraz and Rigaut, Supra, 2006 discloses flow-through mixing devicesconsisting of a conduit through which the lysis solution flows, and aninlet, such as a nozzle, into the conduit in which the cell suspensionis injected in either a counter-flow or co-flow direction.

Brooks, Supra, 2004 claims the use of fluidic vortex mixers forcontinuous flow-through lysis and neutralization. A vortex mixer isdescribed by the patent as a cylindrical chamber with an axial outlet atthe center of one end wall with two tangential inlets along theperiphery. The dimensions of the mixer and the flowrates used are chosenso that the residence time in the mixers is much less that the timerequired for lysis (about 0.01-0.1 sec) so that the cell suspension andlysis solution are mixed completely. The cells can then react with thelysis solution after exiting the vortex mixer. In an example, the cellsand lysis solution are mixed in a first vortex mixer, flow into a tankfor completion of lysis, and then the mixture flows through an outlet ofthe tank where it is mixed with neutralization buffer in a second vortexmixer.

Blanche F, Couder M, Maestrali N, Gaillac D, Guillemin T. 2005 WorldPatent Application WO2005026331 discloses continuous alkaline lysisthrough the use of T tubes with lengths of turbulent flow (achieved bysmall diameter tubing) to rapidly mix the cell suspension and lysissolution, followed by a length of laminar flow (in larger diametertubing) for incubation and time for lysis and denaturation withoutsubstantial agitation which would damage the plasmid and fragment gDNA.Neutralization solution may then be introduced continuously in a secondT tube.

The above flow-through mixing devices enable low shear mixing. It hasbeen generally recognized that shear forces created by mixing toointensely may cause damage to pDNA and fragmentation of gDNA, leading toco-purification of gDNA with pDNA (Horn N A, Meek J A, Budahazi G,Marquet M. 1995 Hum. Gene Ther. 6: 565-573).

Alternative Lysis Methods

Heat Lysis

Plasmid isolation using heat lysis was first reported by Holmes D S,Quigley M. 1981 Anal Biochem 114:193-7, and is perhaps the most widelyused method after alkaline lysis.

Merck has developed and patented processes to adapt heat lysis to largescale processing. In Lee, A L, Sagar, S. 2001 U.S. Pat. No. 6,197,553, abacterial suspension in modified STET buffer (e.g. 50 mM Tris, 50-100 mMEDTA, 8% sucrose, 2% Triton X-100, pH 8.0-8.5) with a density of about30 OD₆₀₀ is pumped through a heat exchanger at such a rate that thesuspension exits with a temperature of 70-100° C., resulting in lysis.The lysate is then centrifuged to pellet large cell debris, protein, andgDNA, leaving RNA and plasmid in solution. The optional use of lysozymeis reported to increase the plasmid concentration in the lysate by 4-5times. It was also determined that the formation of undesirable opencircle plasmid by endogenous DNase during this lysis process could bereduced by increasing the EDTA concentration from 50 mM to 100 mM. Theyreport higher plasmid recovery than by chemical lyses.

A similar process is described by Zhu K, Jin H, Ma Y, Ren Z, Xiao C, HeZ, Zhang F, Zhu Q, Wang B. 2005 J Biotechnol. 118: 257-264, whichreports to have made improvements on the heat lysis methods of Holmesand Quigley, Supra, 1981, and Lee and Sagar, Supra, 2001. In thisprotocol, cell paste is resuspended with 10 mM Tris, 50 mM EDTA, pH 8.0to a density of 100 OD₆₀₀ and treated with 0.1M NaCl, 2% Triton X-100,and lysozyme at 37° C. for 20 minutes. The cell suspension is thenpumped through a copper coil immersed in a 70-80° C. water bath with aresidence time of 20 sec, then it enters another copper coil immersed inan ice bath.

Mechanical Disruption

Generally, mechanical disruption of bacteria (e.g. french press,sonication, homogenization, nebulization) for plasmid isolation is seenas unfeasible due to the damage it would cause to the DNA. Jem K J. 2002U.S. Pat. No. 6,455,287 reports that sonication, nebulization, andGaulin Mill homogenization resulted in almost complete destruction ofpDNA. However, disruption with a bead mill device under optimizedconditions resulted in over 90% of the plasmid solubilized withoutdestruction. They also report that an impinging-jet homogenizer releasedup to 50% of the pDNA intact.

Another method used to overcome destruction of DNA during mechanicaldisruption is the use of DNA compaction agents. Wilson R C, Murphy J C.2002 US Patent Application US2002197637 disclose the use of polycationiccompaction agents (e.g. polylysine, spermine, spermidine) to protect DNAfrom shear damage during mechanical lysis. The compaction agents causethe DNA to be pelleted with the insoluble cell debris. The pellet iswashed, and the pDNA is resolubilized to give an enriched solution. Theuse of compaction agents also results in reduced lysate viscosity.

Lysozyme Lysis

A process developed by Merck (Boyd D B, Kristopeit A J, Lander R J,Murphy J C, Winters M A. 2006 World Patent Application WO2006083721)describes a STET/lysozyme lysis performed at 20° C. or 37° C.,preferably with an additional alkaline pH shift to denature gDNA. Whilethe process retains the pH shifting of alkaline lysis, shear forces arereduced by performing the shift after lysis. Therefore this process doeseliminate many of the difficult processing and equipment needs ofalkaline or heat lysis. The limitation of this reduced temperature lysismethod is the need for large amounts of recombinant lysozyme.

Autolysis

Autolytic strains using phage T4 lysis proteins have been patented forprotein production as in Leung W S, Swartz J R. 2001 U.S. Pat. No.6,258,560. In this system, lysozyme (endolysin) is expressed by the cellin the cytoplasm and released to the periplasm at the desired time byco-expression of a holin (membrane spanning peptide or protein) thatcreates a channel, allowing leakage of lysozyme from the cytoplasm tothe periplasm. Other autolytic E. coli strains that are described in JiaX, Kostal J, Claypool J A. 2006 US Patent Application US20060040393contain the bacteriophage λR lytic endolysin gene. The endolysin isinduced by arabinose, which then causes the E. coli to be lysed after afreeze-thaw cycle.

Autolysis conditions, as opposed to alkaline or heat lysis, do notselectively denature gDNA. The product of lysis is very viscous due tohigh levels of residual gDNA, creating processing problems. For proteinproduction, non specific nucleases (e.g., Benzonase®) are added, orexpressed periplasmically in the strain [e.g., endA or Staphylococcusnuclease (Cooke G D, Cranenburgh R M, Hanak J A J, Ward J M. 2003 JBiotechnology 101: 229-239)] to reduce viscosity after cell lysis. Suchsystems could not be utilized for plasmid production, since the plasmidwould be degraded or damaged by the nuclease. While autolysis is not anessential design improvement for protein production (since cell lysis isperformed at high density using generally available equipment) it hastremendous potential for plasmid purification since alkaline or heatlysis steps are key bottlenecks in plasmid processing.

Bacteriophage T5 exonuclease is an ideal DNase to use in plasmidprocessing. T5 exonuclease does not digest supercoiled plasmid, but isable to digest linear single- and double-stranded DNA (ssDNA, dsDNA). Itwill also digest DNA with denaturation loops, such as ‘ghost’ or ‘shadowband’ DNA, which often retains biological activity and is refractile torestriction enzyme digestion. Williams J A, Hodgson C P. 2006 WorldPatent Application WO2006026125 describe plasmid purification using E.coli strains expressing plasmid-safe nuclease (chimeric ribonuclease-T5exonuclease genes) in combination with endolysin/holin pairs forautolysis.

Cell Disruption Summary

While the basic methods for obtaining plasmids (by bacterialfermentation), and for their purification (e.g., by heat or alkalinelysis) are well-known, and large scale manufacturing methods have beendeveloped, these processes are problematic for transfer to newfacilities due to specialized equipment needs, scaling issues, andtremendous lysis volumes. As well, they add excessive additional cost tothe production of plasmids through reduced capacity, increasedwastestreams, and expensive equipment and reagents. These limitationsplace a cost burden on commercialization of pDNA production processes. Anew technology is needed to eliminate this critical processingbottleneck.

DISCLOSURE OF THE INVENTION

One embodiment of the invention is methods for production of plasmidDNA, using host strains in which a peptidoglycan hydrolase gene isinserted into the bacterial genome and expressed during production.After a plasmid or DNA replicon is grown in the cells, the peptidoglycanhydrolase is released from the cytoplasm, digesting the bacterial cellwall.

In one preferred embodiment, autolysis, peptidoglycan hydrolasedigestion of the cell wall is utilized to effect cell lysis (autolysisprocess). In a preferred embodiment, peptidoglycan hydrolase expressingcells are used as an endogenous source of lysozyme in standard heat orlysozyme/STET lysis methods. In a preferred embodiment, cell lysis ismodified from standard alkaline or heat lysis methods to lysis with asingle solution containing concentrations of flocculating agents andsalts that allows: 1) plasmid to be released from the cells and becomesoluble in the lysis solution and; 2) bacterial gDNA removal by itsinsolubility in the lysis buffer. In a preferred embodiment, theresulting liquid lysate containing pDNA is purified in a downstreamprocess. In a preferred embodiment, after removal of cell debris andinsoluble gDNA and other impurities, the pDNA is recovered byprecipitation through the addition of more salt.

In an alternative preferred embodiment, extraction, peptidoglycanhydrolase digestion of the cell wall is utilized to permeabilize cellsto affect plasmid release without complete lysis of the cell orextensive extraction of gDNA. In one preferred embodiment, theextraction process is performed by resuspension of peptidoglycanhydrolase containing cells in a buffer containing Triton X-100 and EDTAand/or polyethyleneimine at a slightly acid pH (low pH extractionprocess). In a preferred embodiment, extracted cells are flocculated byheat treatment and removed by filtration. In a preferred embodiment,plasmid present in the liquor after removal of extracted cells ispurified in a downstream process.

In one preferred embodiment, the peptidoglycan hydrolase is abacteriophage endolysin. In another preferred embodiment, the endolysinis the bacteriophage lambda gene R protein. In yet another preferredembodiment, the endolysin is the bacteriophage T4 gene e protein. In yetanother preferred embodiment, the endolysin is the bacteriophage T7lysozyme protein. In yet other preferred embodiments, the endolysin is acombination of endolysins, such as phage T4 (gene e) and phage lambda(lambdaR) endolysin proteins.

In yet another preferred embodiment, methods for producing peptidoglycanhydrolase in cells are disclosed, using host strains in which apeptidoglycan hydrolase gene is inserted into the bacterial genome or Fplasmid, and selectively expressed during production. In a preferredembodiment the integrated peptidoglycan hydrolase is cloned downstreamof a heat inducible promoter and selectively expressed during productionby heat induction. In yet another preferred embodiment, the heatinducible promoter is the bacteriophage lambda pR and/or pL promoter(s)regulated by the C1857 temperature sensitive lambda repressor.

In yet another preferred embodiment, methods for improving fermentationyield and quality are disclosed, wherein fermentation cells are held atreduced temperature post production. In a preferred embodiment, theculture is held at 25-30° C. for 0.5 to 2 hrs post plasmid productionprior to harvesting.

In yet another preferred embodiment, methods for improving plasmid yieldand cell line viability are disclosed, wherein bacterial seed stocks aremade at reduced temperature prior to cryopreservation. In a preferredembodiment, seed stocks are manufactured by growth of cells at 25-30° C.during production.

BRIEF SUMMARY OF THE INVENTION

It is a purpose and/or objective of the present invention to providecompositions of matter and processes for plasmid production. Anotherobjective of the invention is to provide methods to reduce nucleic acidimpurities, such as RNA and genomic DNA, in purified pDNA. Yet anotherobjective of the invention is to reduce production costs for pDNApurification. Yet another objective and/or purpose of the invention isto reduce toxic waste in pDNA purification. Another objective of theinvention is improved plasmid production processes that, compared toprocesses defined in the art, are improved by: increased quality ofplasmid by reduced levels of nicked (open circular) or linearizedversions of the plasmid; simplified production using robust productionsteps in dramatically reduced process volumes; simplified productionthrough elimination of multiple production steps; reduced cost throughelimination of multiple production steps and reduction of processvolumes; reduced cost through elimination of expensive reagentscurrently utilized in cell lysis; increased quality of plasmid byreduction of nucleic acid impurities after plasmid purification due toelimination of key contaminants prior to entry into downstreamprocessing; improved regulatory compliance by elimination of gDNA fromfinal plasmid preparations; and improved regulatory compliance byelimination of toxic waste streams.

Further objects and advantages of the invention will become apparentfrom a consideration of the drawings and ensuing description.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1. Plasmid production flowchart

FIG. 2. Application of various embodiments of the invention in plasmidproduction

FIG. 3. Plasmid extraction

FIG. 4. Overview of the inducible fed-batch fermentation process

FIG. 5. Plasmid quality analysis of samples from an inducible fed-batchfermentation.

FIG. 6A. pAHI44 heat inducible overexpression vectors

FIG. 6B. pAHI44 heat inducible overexpression vectors

FIG. 6C. pAHI44 heat inducible overexpression vectors

FIG. 7. STET cell suspensions

FIG. 8. Agarose gel analysis of clarified lysates

FIG. 9. Agarose gel analysis of PEG clarified lysates

FIG. 10. Agarose gel analysis of PNL clarified lysates

FIG. 11. Agarose gel analysis of samples from a purification of pDNAusing autolysis and filter membranes

FIG. 12. Agarose gel analysis of samples from a purification of pDNAusing autolysis and a non-chromatographic process

FIG. 13. Effects of salt concentration, extraction time, Triton X-100,and PEG8000 on plasmid extraction from autolytic cells

FIG. 14. Effect of pH and sodium acetate concentration on plasmidextraction. Agarose gel analysis

FIG. 15. low pH extraction of various plasmids, with or withoutendolysin, is shown

FIG. 16. Substitution of lysozyme for endolysin in low pH extraction isshown

FIG. 17A. Improved solid liquid separation by thermal flocculation of anautolytic extraction mixture

FIG. 17B. Improved solid liquid separation by thermal flocculation of anautolytic extraction mixture

FIG. 18. Improved plasmid quality by thermal flocculation of anautolytic extraction mixture

Table 1: Process hold step post 42° C. increases plasmid yield

Table 2: Seed stock preparation at reduced temperature increases plasmidyield

Table 3: Autolysis using Endolysin expressing cell lines

Table 4: Extraction of pDNA from endolysin containing cells usingacetate solutions

Table 5: Protein extraction

Table 6: EDTA versus PEI extraction efficiency

Table 7: SDS-Calcium conditioning of a plasmid extract to remove RNA

Table 8: Thermal Treatment

DETAILED DESCRIPTION OF THE INVENTION

Turning now to the drawings, in FIG. 1, a flowchart of plasmidproduction is shown.

In FIG. 2, the plasmid production flowchart is modified to highlightimprovements to specified steps by various embodiments of the invention.

In FIG. 3, the structure of an E. coli cell is shown, highlightingvarious barriers to plasmid extraction, and example chemical or enzymetreatments that permeabilize each barrier.

In FIG. 4, the inducible fed-batch fermentation of Carnes and Williams,Supra, 2006 is shown, in which plasmid-containing E. coli cells aregrown at a reduced temperature during part of the fed-batch phase,during which time growth rate is restricted, followed by a temperatureup-shift and continued growth at elevated temperature in order toaccumulate plasmid.

In FIG. 5, Plasmid quality analysis over the course of an induciblefed-batch fermentation is shown. Plasmid production was induced from 30°C. to 42° C. at 25 hrs. Induction was for 14 hrs (25-39 hrs) followed bya 2 hr hold at 25° C. The ratio of supercoiled to open-circle plasmidwas determined with the PlasmidSelect Xtra Screening Kit (GE Healthcare,Uppsala, Sweden). The supercoiled plasmid purity in the final sample (41hours) after a 2 hr hold at 25° C. is 96%.

In FIG. 6, maps of the pAH144 zwf-lambdaR heat inducible overexpressionvector, and selected precursors, are shown.

In FIG. 7, three STET cell suspension aliquots after aging 30 minutes atroom temperature are shown.

In FIG. 8, agarose gel analysis of clarified lysates using lysissolutions containing glucose and/or Triton X-100 and EDTA are shown.Endolysin containing fermentation cells carrying a high copy DNA vaccineplasmid were utilized in autolysis. Lanes: M) 1 kb DNA ladder(Invitrogen, Carlsbad Calif.); 1) a miniprep sample of the plasmid; 2)autolysis with 0.04% Triton X-100, 1 mM EDTA, 0% glucose; 3) autolysiswith 0.04% Triton X-100, 50 mM EDTA, 0% glucose; 4) autolysis with 0.04%Triton X-100, 1 mM EDTA, 8% glucose; 5) autolysis with 0.04% TritonX-100, 50 mM EDTA, 8% glucose; 6) autolysis with 2% Triton X-100, 1 mMEDTA, 0% glucose; 7) autolysis with 2% Triton X-100, 50 mM EDTA, 0%glucose; 8) autolysis with 2% Triton X-100, 1 mM EDTA, 8% glucose; 9)autolysis with 2% Triton X-100, 50 mM EDTA, 8% glucose.

In FIG. 9, agarose gel analysis of clarified lysates using lysissolution containing PEG and different amounts of NaCl are shown.Endolysin containing fermentation cells carrying a high copy DNA vaccineplasmid were utilized in autolysis. Lanes: M) 1 kb DNA ladder; 1)Standard: 200 ng purified plasmid; 2) Supernatant of autolysis with 7.5%PEG-8000, 50 mM Tris pH 8.0, 10 mM EDTA, 1% Triton X-100; 3) Supernatantof autolysis with 7.5% PEG-8000, 50 mM Tris pH 8.0, 10 mM EDTA, 1%Triton X-100+0.1M NaCl; 4) Supernatant of autolysis with 7.5% PEG-8000,50 mM Tris pH 8.0, 10 mM EDTA, 1% Triton X-100+0.2M NaCl; 5) Supernatantof autolysis with 7.5% PEG-8000, 50 mM Tris pH 8.0, 10 mM EDTA, 1%Triton X-100+0.3M NaCl; 6) Supernatant of autolysis with 7.5% PEG-8000,50 mM Tris pH 8.0, 10 mM EDTA, 1% Triton X-100+0.4M NaCl; 7) Same aslane 6; 8) TE (10 mM Tris pH 8.0, 1 mM EDTA) extraction of insolublematerial pelleted from autolysis with 7.5% PEG-8000, 50 mM Tris pH 8.0,10 mM EDTA, 1% Triton X-100+0.4M NaCl

In FIG. 10, agarose gel analysis of clarified lysate by autolysis in PNLbuffer with SDS and Ca²⁺ is shown. Endolysin containing fermentationcells carrying a high copy DNA vaccine plasmid were utilized inautolysis.

In FIG. 11, agarose gel analysis of samples from a purification of pDNAusing autolysis and filter membranes is shown. Endolysin containingfermentation cells carrying a high copy DNA vaccine plasmid wereutilized in autolysis. Lanes: M) 1 kb DNA ladder; 1) clarified lysate,2) filtrate of the plasmid precipitation mixture; 3) wash filtrate; 4)first 5 mL TE filtrate fraction; 5) second 5 mL TE filtrate fraction; 6)75 mL TE filtrate fraction.

In FIG. 12, agarose gel analysis of samples from a purification of pDNAby autolysis and a non-chromatographic process is shown. Endolysincontaining fermentation cells carrying a high copy DNA vaccine plasmidwere utilized in autolysis. Lanes: M) 1 kb DNA ladder; 1) 2 μL clarifiedlysate; 2) 2 μL clarified lysate after 65° C. step; 3) 2 μL supernatantafter precipitation of plasmid DNA by NaCl addition; 4) 2 μL of theredissolved plasmid DNA; 5) 2 μL after removal of the hydrated calciumsilicate; 6) 0.1 μL of the final plasmid solution (diluted prior toloading).

In FIG. 13, effects of salt concentration, extraction time, Triton X-100and PEG8000 on plasmid extraction from endolysin containing fermentationcells carrying a high copy plasmid are shown. Lanes: M, 1 kb DNA ladder;C, 0.5 μg of the same plasmid prepared by alkaline lysis (equivalent100% recovery for comparison of the extractions shown). 1 μL ofsupernatant from each of the extractions described in Table 4 (samples1-10A, B or C) was loaded.

In FIG. 14, the effect of pH and sodium acetate concentration on plasmidextraction is shown. Agarose gel analysis, Lanes: M, 1 kb DNA ladder; 1,0.5 μg plasmid control sample; A lanes, 0.4 M sodium acetate; B lanes,0.6 M sodium acetate; C lanes, 0.8 M sodium acetate, D lanes, 1.0 Msodium acetate.

In FIG. 15, low pH extraction of various plasmids, with or withoutendolysin, is shown.

In FIG. 16, substitution of lysozyme for endolysin in low pH extractionis shown.

In FIG. 17, improved solid liquid separation by thermal flocculation ofan autolytic extraction mixture is shown. A) Autolytic acidic plasmidextraction lysate followed by thermal treatment resulting inflocculation and sedimentation of cell debris. B) Lysate prepared fromautolytic cell paste in a pH 8.0 buffer resulting in poor flocculationand sedimentation of cell debris.

In FIG. 18, an agarose gel analysis of lysates prepared by either thelow pH extraction—thermal flocculation process, or by pH 8 heat lysisare shown. Lanes: M, 1 kb DNA ladder; 1, 1 μg DNA from low pHextraction—thermal flocculation process; 2, 1 μg DNA from the pH 8 heatlysis process.

DEFINITIONS

Amcyan: Anemonia majano cyan fluorescent protein.

autolysin: Bacterial encoded endolysin-like protein that can mediateautolysis.

autolysis: Lysis methods that cause the cell to undergo self lysis, suchas β lactam induced cell lysis, autolysin induced lysis, phiX174 phagelysis protein induced ghosting, T4 or lambda phage induced cell lysisby: phage lysozyme/phage holin coexpression; freeze thaw, or; phagelysozyme in combination with buffers that permeabilize the innermembrane, etc.

autolysis process: Peptidoglycan hydrolase digestion of the cell wall isutilized to effect cell lysis.

autolyte buffer: Low pH extraction buffer of the composition 30 mMsodium acetate, 50 mM EDTA, 8% sucrose, 0.1% Triton X-100, pH 5.2; ormodifications that include up to 400 mM sodium acetate, 0-50 mM EDTA0-100 μg/mL PEI, pH 4.7-5.5, 0.05-2% Triton X-100 or 0.5-1% SDSsubstituted for Triton X-100.

ccc: Covalently Closed Circular.

DNA replicon: Plasmids, cosmids, bacterial artificial chromosomes(BACs), bacteriophages, viral vectors and hybrids thereof.

E. coli: Escherichia coli, a gram negative bacteria

EDTA: ethylene diamine tetraacetic acid.

EGFP: enhanced green fluorescent protein.

endolysin: Phage encoded lysozyme, such as those encoded by thebacteriophage lambda gene R (λR), bacteriophage T4 gene e, andbacteriophage T7 lysozyme genes.

EU: Endotoxin units, KEU=kilo endotoxin units, a measurement ofLipopolysaccharide (LPS) content.

g: Gram, kg for kilogram, mg for milligram.

gDNA: Genomic DNA.

ghost band: Denatured ccc DNA.

HA: Hemagglutinin.

holin: Phage protein that inserts into the cytoplasmic membrane andforms pore allowing endolysin access to cell wall.

HEWL: Chicken (hen) egg white lysozyme.

Hr(s): Hour(s).

Kd: Kilodalton.

L: Liter, mL for milliliters, μL for microliters.

low pH extraction process: Peptidoglycan hydrolase digestion of the cellwall is utilized to permeabilize cells to effect plasmid release withoutcomplete lysis of the cell or extensive extraction of gDNA byresuspension of peptidoglycan hydrolase containing cells in a low pHautolyte buffer or equivalent (e.g. citrate based buffer).

min: Minute.

OD₆₀₀: Optical density at 600 nm.

OD-unit: One OD-unit is equivalent to the amount of cells, which whensuspended in a volume of 1.0 mL, gives an OD₆₀₀=1.0.

pDNA: Plasmid DNA.

PEG: Polyethylene glycol.

PEI: Polyethyleneimine.

peptidoglycan hydrolase: Murein hydrolases, including endolysin,autolysins or lysozymes that digest the cell wall (murein). Reviewed inSalazar O, Asenjo J A 2007 Biotechnol. Lett 29:985-994.

phage lysis proteins: Proteins cause the cell to undergo self lysis orghosting such as ghosting induced by phiX174 phage lysis protein, orcell lysis induced by T4 or lambda phage lysozyme-holin coexpression.

plasmid: Plasmids, cosmids, bacterial artificial chromosomes (BACs),bacteriophages, viral vectors and hybrids thereof.

plasmid extraction: Endolysin digestion of the cell wall is utilized ina process to permeabilize cells to effect plasmid release withoutcomplete cell lysis (endolysin mediated non-lytic cellpermeabilization).

plasmid-safe nuclease: Exonuclease such as T5 exonuclease that degradesvarious forms of DNA but not covalently closed circular (ccc) DNAincluding pDNA.

PNL buffer: A solution containing approximately 7.5% PEG-8000, 1-100 mMEDTA, 10-50 mM Tris pH 7-9, and salt in the range of 0-1 M.

pNTCultra: pDNAVACCUltra DNA vaccine plasmids as disclosed in Williams,J A 2006 World Patent Application WO2006078979, with various backbonemodifications or antigen genes.

precipitant: A substance that causes a precipitate to form when added toor included in a solution.

precipitate: n. A solid or solid phase separated from a solution. v. Tobe separated from a solution as a solid.

RBS: Ribosome Binding Sites.

RNase: Ribonuclease.

RNaseA: Bovine pancreatic ribonuclease A.

RT-PCR: Real time PCR.

SDS: Sodium dodecyl sulphate, an anionic detergent.

SDS-Calcium: SDS-CaCl₂, a method of forming a flocculant precipitateafter sequential addition of a SDS anionic detergent component followedby a calcium chloride (CaCl₂) cation component. This forms CalciumDodecyl Sulfate, a calcium surfactant that forms insoluble flocculatedcomplexes (calcium dodecyl sulfate complexes) with protein,lipopolysaccharides, and other host cell impurities while leaving pDNAin solution.

sec: Seconds.

STET buffer: A solution containing approximately 8% sucrose or 8%glucose, 0.01-5% Triton X-100, 10-100 mM EDTA, 10-50 mM Tris pH 8.0.

synthetic anionic surfactant: Formed after sequential addition of aanionic detergent component followed by a cationic component, preferablyan alkaline earth metal (e.g. Calcium Dodecyl Sulfate, a calciumsurfactant formed by sequential addition of SDS-CaCl₂).

T5 exonuclease: Bacteriophage T5 D15 exonuclease.

TE buffer: A solution containing approximately 10 mM Tris pH 8 and 1 mMEDTA.

TFF: Tangential flow filtration or cross flow filtration.

Tris: trishydroxymethylaminomethane. Stock solutions are typically madeusing either Tris-base, pH adjusted down with hydrochloric acid (HCl) orwith Tris-HCl, pH adjusted up with sodium hydroxide (NaOH).

WCW: Wet cell weight.

zwf: Glucose 6-phosphate dehydrogenase.

The invention relates to methods for the production and purification ofplasmid DNA (pDNA) using the gram negative bacterium E. coli as aproduction host. One embodiment of the invention includes alternativecell lysis and plasmid extraction methodologies that utilize endolysinproducing host strains. These methodologies eliminate alkaline,lysozyme, or heat lysis methodologies which are by nature problematicand are a major process bottleneck (FIG. 1). A summary of application ofvarious embodiments of the invention is shown in FIG. 2.

Another major problem of purification technology has been the separationof pDNA from E. coli gDNA. Some embodiments of the invention disclosemethods for reducing gDNA during isolation of covalently closed circular(ccc) DNA. This is accomplished, for example, through PEG flocculationof an autolysate which removes cell debris as well as gDNA. In analternative embodiment, gDNA is reduced by extracting plasmid from cellsunder conditions in which gDNA is retained in the cells.

Autolysis Process Preferred Embodiments

Cells are produced in fermentation culture. After production, plasmid ispurified from the cells.

In a preferred embodiment, cells are lysed using autolysis. A preferredmethod of autolysis is to disrupt cells by release of cytoplasmicpeptidoglycan hydrolase that is produced by the host strain. In apreferred embodiment, the peptidoglycan hydrolase is a bacteriophageendolysin. In a preferred embodiment, endolysin is expressed in thecytoplasm of the cell and released from the cytoplasm after cell growthby utilization of a lysis buffer that permeabilizes the inner cellmembrane. This is superior to processes that utilize: 1) holin geneexpression to release endolysin from the cytoplasm, or; 2) freeze thawto release endolysin from the cytoplasm or; 3) recombinant lysozyme todegrade the cell wall. Holin gene expression during growth to releaseendolysin does not allow cell harvest or storage, or the control of thetime and buffer composition of lysis. Freeze thaw is not a scaleablemanufacturing process. Recombinant lysozyme adds prohibitive cost toplasmid production (Hoare M, Ley M S, Bracewell D G, Doig S D, Kong S,Titchener-Hooker N, Ward J M, Dunnill P. 2005 Biotechnology Progress.21: 1577-1592).

The application of peptidoglycan hydrolase expressing cell lines toplasmid production is not taught in the art. A number of investigatorshave developed plasmid isolation processes that utilize lysozyme. Theseinclude methods for lysing EDTA-lysozyme spheroplasts based on theoriginal description of Clewell D B 1972 J Bacteriol. 110: 667-676 andClewell D B, Helinski D R. 1970 Biochemistry 9:4428-4440. Boyd et al,Supra, 2006 discloses a large scale STET/recombinant lysozyme lysismethod for plasmid production. These inventors do not contemplateutilization of autolytic cells to replace the need for exogenouslysozyme. Lysozyme is also utilized in the large scale heat lysisprocess of Lee and Sagar, Supra, 2001. These inventors also do notcontemplate utilization of autolytic cells to replace the need forexogenous lysozyme as is disclosed herein.

A number of investigators have developed protein isolation processesthat utilize endolysin cell lines. Crabtree S, Cronan J E. 1984 JBacteriol. 158: 354 disclose use of pJH2 (inducible lambda R and Rzendolysin, with S gene mutation, so endolysin only) to lyse E. colistrains by freeze thaw. The disclosure teaches using autolysis for celllysis, and does not contemplate using autolysis for plasmid or DNApurification. Leung and Swartz, Supra, 2001 disclose autolytic strainsusing arabinose inducible phage T4 lysis proteins. In this system,lysozyme (T4 gene e endolysin) is expressed by the cell in the cytoplasmand released to the periplasm at the desired time by co-expression of aholin (membrane spanning peptide or protein) that creates a channel,allowing leakage of lysozyme from the cytoplasm to the periplasm. In analternative embodiment, T4 endolysin is used without holin to lyse cellsafter a freeze thaw cycle. This disclosure teaches that autolysisrequires either holin gene expression or freeze thaw for lysis. Thisdisclosure also teaches using autolysis for protein purification, anddoes not contemplate using autolysis for plasmid or DNA purification.Auerbach J, Rosenberg M, 1987 U.S. Pat. No. 4,637,980, disclose lysogenswhich contain single copy, inducible lambda endolysin, for cell lysis.The disclosure indicates in theoretical terms that the strains can beutilized for plasmid purification, but data to support this is notdisclosed in the application. Other autolytic E. coli strains taught byJia et al, Supra 2006 contain the bacteriophage λR lytic endolysin geneintegrated into the genome. The endolysin is induced by arabinose, whichthen causes the E. coli to be lysed after a freeze-thaw cycle. Thisdisclosure teaches that autolysis with endolysin protein containingcells requires freeze thaw lysis; multiple examples are listed and inall cases freeze thaw was required for autolysis. This teaches away fromthe current disclosure of endolysin mediated autolysis in simple buffersolutions without freeze thaw. The disclosure indicates in theoreticalterms that the strains can be utilized with freeze thaw for nucleic acidextraction and isolation, but data on nucleic acid extraction is notdisclosed in the application. Studier F W. 1991, J Mol. Biol. 219: 37-44disclose cell lines containing the pLysS or pLysE plasmids encodingconstitutively produced cytoplasmic phage T7 lysozyme; theses cell lineshave been utilized by dozens of investigators to lyse cells for proteinisolation using one or more freeze thaw cycles or Triton X-100/EDTAlysis solutions, but have not been applied to plasmid purification.

Kloos D, Stratz M, Guttler A, Steffan R J, Timmis K N. 1994 J Bacteriol.176: 7352-7361, and Jain V, Mekalanos J J. 2000 Infect Immun 68: 986-989disclose endolysin/holin (lambda S, R and Rz) expressing plasmids thatare used to lyse cells and release DNA. Use of these systems for pDNApurification are not contemplated by the authors, and the systems arenot applicable for purification of heterologous plasmids since suchplasmid preparations would be contaminated with the lysis gene plasmid(the lysis genes are not integrated). As well, the systems utilize holingenes (Lambda S) for lysis.

Williams and Hodgson, Supra, 2006 disclose the use of endolysin/holincombination expressing cell lines for plasmid production, and the use ofplasmid safe exonuclease to reduce viscosity after lysis. The authors donot disclose the use of endolysin only cell lines for plasmidproduction.

We disclose herein the application of endolysin expressing cell lines toplasmid production. The disclosed autolysis process using the endolysinexpressing cell line embodiments of the invention is a critical designimprovement in bacterial cell lysis. As discussed above, current lysismethods for plasmid production require addition of expensive lysozyme(e.g. Boyd et al, Supra, 2006; Zhu et al, Supra, 2005). This is not anoptimal manufacturing process, due to cost and regulatory concern(lysozyme). As well, endolysin-holin lysis systems have scaling andprocess control issues. In-fermentor holin lysis destroys cells prior tocell testing or release by Quality Assurance and prevents normal harvestunit operations to be performed. The endolysin mediated autolysisprocess disclosed herein is a scaleable cost effective process thatmeets a long felt, but unsolved need for elimination of alkaline or heatlysis methodologies which, as discussed previously, are problematic dueto specialized equipment needs, scaling issues, and tremendous lysisvolumes.

Preferred peptidoglycan hydrolase genes for practicing variousembodiments of the invention are chicken (hen) egg white lysozyme(HEWL), alternative lysozymes, autolysins, or bacteriophage endolysingene products. Preferred endolysin genes are the bacteriophage lambda R,the bacteriophage T4 gene e, or the bacteriophage T7 lysozyme genes.Alternative peptidoglycan hydrolase genes that may be utilized topractice various embodiments of the invention, and a discussion of theiraction, are disclosed in Salazar and Asenjo, Supra, 2007 and areincluded herein by reference.

Extraction Process Preferred Embodiments

In a preferred embodiment, plasmid is extracted from cells withoutcomplete cell lysis. A preferred method of plasmid extraction is todisrupt cells by release of cytoplasmic endolysin that is produced bythe host strain and permeabilize the cell wall under conditions inwhich: 1) the cell does not completely lyse, and 2) plasmid is releasedfrom the cell. In a preferred embodiment the conditions in which, 1) thecell does not completely lyse, and 2) plasmid is released from the cell,comprise low pH treatment (pH 4.7-6.9) of endolysin containing cellswith nonionic or ionic detergents (low pH extraction process). In apreferred embodiment, the low pH extraction process is used to extract asoluble protein from the cell. The inventors have surprisinglydetermined that this can be accomplished by permeabilizing the innermembrane of an endolysin cell line, or by permeabilizing the innermembrane and outer membrane of an non-endolysin cell line in thepresence of lysozyme (FIG. 3). Optionally, outer membrane permeabilizingcompounds such as EDTA are included to improve the yield of extractedplasmid DNA with endolysin expressing cell lines.

A preferred composition of the extraction solution contains 0.03-0.4 Msodium acetate, 0-50 mM EDTA, 0-8% sucrose pH 4.7-6.0 in which thenonionic detergent is 0.05-1% Triton X-100. Yet another preferredcomposition of the extraction solution contains 0.03-0.4 M sodiumacetate, 0-50 mM EDTA, 0-8% sucrose pH 4.7-6.0 in which the ionicdetergent is 0.5-1% SDS. Yet another preferred composition of theextraction solution contains 0.03M sodium acetate, 50 mM EDTA, 0-8%sucrose pH 4.8-5.2 in which the nonionic detergent is 0.05-1% TritonX-100. Yet another preferred composition of the extraction solutioncontains 0.03M sodium acetate, 50 mM EDTA, 8% sucrose pH 5.0 in whichthe ionic detergent is 0.5-1% SDS. Yet another preferred composition ofthe extraction solution contains 0.4M sodium acetate, 10 mM EDTA, pH4.8. Yet another preferred composition of the extraction solutioncontains 0.03M sodium acetate, 50 mM EDTA, 8% sucrose pH 5.0.Alternative outer membrane-permeabilizing compounds, such aspolyethyleneimine (Helander I M, Alakomi H L, Latva-Kal K, Koski P.1997. Microbiology 143:3193-3199) may also be included, in addition to,or replacing EDTA. Yet another preferred composition of the extractionsolution contains 0.03M sodium acetate, 1 mM EDTA, 50 μg/mLpolyethyleneimine, 8% sucrose, pH 5.2 in which the nonionic detergent is0.05-0.1% Triton X-100. Various ionic and or nonionic detergents arealso contemplated for use in place of Triton X-100. For example, variouscompounds that permeabilize the outer membrane are discussed in Vaara M.1992 Microbiological Reviews 56: 395-411 and Leung and Swartz, Supra,2001, and are included herein by reference.

Resuspension of cells in these solutions results in extraction of pDNAwithout substantial extraction of gDNA or cell lysis, or excessiveviscosity. The inventors disclose herein that other salts or acids (e.g.potassium acetate, lithium acetate, sodium citrate, acetic acid, citricacid) may also be used in this process. Additional preferred componentsand concentrations can be determined by one skilled in the art.Autolytic cells containing pDNA can be resuspended or buffer exchangedwith the extraction solution to recover pDNA. The inventors discloseherein that pDNA extraction occurs quickly upon resuspension ofconcentrated autolytic cells or autolytic cell paste in the extractionsolution.

The use of endolysin expressing strains in the plasmid extraction methodembodiments of the invention is a critical design improvement inextraction of pDNA from cells. Chemical methods for non-lyticpermeabilization of bacterial cells have been described in the art.Genomic DNA release from E. coli has been reported in the presence ofEDTA and low ionic strength (i.e. TE buffer). Inclusion of salt reducedDNA release, as did elimination of EDTA (Paul J H, David A W. 1989 Appl.Environ. Micro. 55: 1865-1869). Subsequently, leakage of low levels ofplasmid from cells under similar low ionic conditions has been reported.Plasmid leakage was observed in aqueous solutions, for example 10 mMTris 10 mM EDTA, pH 8.5, water, 10 mM Tris/1% Tween 20. Yield was low,and much of the released plasmid was nicked or linearized. Addition ofprotease K was required to obtain significant amounts of plasmidleakage, and much of the released plasmid remained nicked or linearized(Baker M, Taylor M. 2003 World Patent Application WO03046177). Althoughacceptable for analytical sample preparation, nicked plasmid isdifficult to remove; as well, enzyme treatments are not economical inlarge scale manufacture. In the case of EDTA permeabilization, EDTA isknown to permeabilize cells at sensitive sites adjacent to divisionsepta; this permeabilization could account for the low yield of plasmidleakage without enzymatic treatment since division septa are present on<1% of stationary phase cells, and only 20-30% of mid-log phase cells(Shellman V L, Pettijohn D E. 1991J Bacteriol. 173:3047-3059).

In another example, harsh conditions, heating the bacteria at 80-95° C.in a permeabilization solution containing a non-ionic detergent (0.07%Triton X-100) a metal chelating agent (10 mM EDTA), and optionally anionic detergent (1% lithium lauryl sulfate) were employed to releaseribosomal RNA and gDNA suitable for analytical applications (Clark K A,Kacian D L. 1998 U.S. Pat. No. 5,837,452). The integrity of released DNAwas not assessed, nor was release of pDNA.

Disclosed herein, the inventors have identified that non-endolysinfermentation production cells are resistant to EDTA or non-ionicdetergent extraction using the conditions of Clark and Kacian, Supra,1998 and/or Baker and Taylor, Supra, 2003. This unexpected observationdemonstrates that non-lytic plasmid extraction methods taught in the artare not applicable to plasmid fermentation cells. We disclose herein thenovel observation that plasmid fermentation cells expressing endolysinare permissive to plasmid DNA extraction in a variety of bufferconditions.

While not limiting the application of the invention embodiments, theresistance of fermentation cells to plasmid extraction may be related tothe observation that E. coli cells vary tremendously in their resistanceto sonication, electroporation (Calvin N M, Hanawalt P C. 1988 J.Bacteriol. 170: 2796-2801), or pressure treatment (Manas P, Mackey B M.2004 Appl Environ Microbiol. 70: 1545-54) depending on the stage ofgrowth or growth temperature. In general, log phase cells are moresensitive, while stationary cells are more resistant to lysis orleakage. However, late stage stationary phase anoxic saturated culturesutilized for reported plasmid leakage studies (e.g. Baker and Taylor,Supra, 2003) are less healthy, and may leak plasmid due to cell membraneweakening in response to anoxia. The unexpected observation that thepresence of endolysin facilitates plasmid extraction from fermentationcells enables the application of plasmid extraction to large scaleplasmid production from fermentation cells.

The use of a slightly acidic solution to extract plasmid from cells,with or without endolysin, is not taught in the art. Rather, the artteaches that endolysin and lysozyme function poorly at low pH.Characterization of the activity of HEWL, T4 endolysin (T4 gene e)(Jensen H B, Kleppe K. 1972, Eur. J. Biochem 28:116-122) and lambda Rendolysin (Evrard C, Fastrez J, Soumillion P. 1999 FEBS letters 460,442-446) demonstrated very little activity of all these enzymes below pH6. Consistent with this, poor protein release and lysis with the Xjacell line (integrated lambdaR endolysin) in a 100 mM sodium acetatebuffer at pH 5, or pH 5.5-6.5 using Tris or phosphate buffers is taughtby Jia, et al, Supra, 2006.

Plasmid purification itself is generally not done at low pH. One heatlysis process has been developed wherein bacteria are captured on afilter cake, and lysed at 70° C. in a pH 4.7 citrate buffer. In thiscase, plasmid is retained on the filter cake after lysis. Lysozyme isutilized, but in a previous neutral pH fragilization step rather than inthe lysis buffers (O'Mahony K, Freitag R, Hilbrig F, Schumacher I,Muller P. 2007 Biotechnol Prog. 23: 895-903). This study does not teachlow pH extraction of plasmid (cells are lysed under conditions in whichplasmid is not released), and teaches away from the current embodimentof the invention as it further teaches that lysozyme will not functionin a low pH lysis buffer.

Homogenization of cells preconditioned at pH 4-4.5 has been reported toimprove cell debris removal after homogenization through reducedbiomass-biomass interaction (Gehart R L, Daignault R A 2004 World PatentApplication WO2004022581). This method is utilized in proteinpurification after homogenization; the authors did not contemplate useof the method without homogenization or to extract pDNA.

The art has demonstrated that treatment of cells with low amounts oflysozyme can be used to isolate “lysozyme nucleoids” in whichcytoplasmic protein and RNA is released while the nucleoid retainslargely intact cell boundaries and DNA (Reviewed in Murphy L D,Zimmerman S B 2001 J Structural Biol. 133: 75-86). These studies do notteach that pDNA is released separate from gDNA. Consistent with this ina plasmid purification process in which lysozyme EDTA spheroplasts areisolated prior to lysis for plasmid purification, plasmid was associatedwith the spheroplasts; this teaches that plasmid is not released bylysozyme-EDTA treatment (Wicks. I P, Howell M L, Hancock T, Kohsaka H,Olee T, Carson D A. 1995 Human Gene Therapy 6: 317-323).

A variety of studies that demonstrated that the majority of pDNA in acell with relaxed replication such as ColE1 is separable from gDNAlikewise did not identify conditions for extracting plasmid (e.g. KlineB C, Miller J R, Cress D E, Wlodarczyk M, Manis J J, Otten M R. 1976. JBacteriol. 127: 881-889).

Autolysis Process Lysate Clarification Preferred Embodiments

The product of the autolysis process, as opposed to the low pHextraction process (which doesn't liberate gDNA) or alkaline or heatlysis (which selectively denatures gDNA) is very viscous, creatingprocessing problems. In one preferred embodiment of the invention,viscosity is reduced by digesting gDNA utilizing a coexpressed plasmidsafe nuclease as described in Williams and Hodgson, Supra, 2006. Inanother preferred embodiment of the invention, viscosity is reduced byflocculating and removing gDNA and cell debris.

A variety of different flocculants can be used to aggregate host celldebris. Several are disclosed in Boyd et al, Supra, 2006 and areincluded herein by reference. In one preferred embodiment of theinvention, gDNA and cell debris are removed by flocculation withpolyethylene glycol (PEG).

By way of example, consider the following methods to purify pDNA withthe PEG lysis process

-   -   1) A preferred composition of the PEG lysis buffer contains        concentrations of PEG and a salt in which pDNA is soluble and        gDNA and/or other impurities are insoluble. For example, a        preferred composition contains 7.5% w/v PEG-8000, and 0.1-0.2 M        NaCl. Additional preferred components and concentrations can be        determined by one skilled in the art, for example, 10 mM EDTA,        50 mM Tris, and 0.05 to 2% Triton X-100.    -   2) A preferred embodiment is the use of this PEG lysis process        for purification of pDNA from autolytic strains. Suspension of        autolytic cells in the PEG lysis buffer lyse readily without        freeze-thaw. This process may also be used with non-autolytic        cells, and addition of lysozyme may be used to aid lysis.    -   3) In another preferred embodiment, protein and other impurities        can be precipitated and removed by heat treatment and        clarification of the PEG lysate. A preferred heat treatment is        performed by heating the lysate to 65° C.-75° C. for at least 15        minutes.    -   4) In another preferred embodiment, pDNA may optionally be        recovered from the lysate after clarification by precipitation.        A preferred method of precipitation of the pDNA from the PEG        lysate is performed by raising the NaCl concentration of the        solution to >0.3M NaCl. Precipitation of pDNA by this method        also results in the removal of RNA, since RNA remains soluble        and will be separated from the precipitated pDNA.    -   5) In a preferred embodiment an ionic detergent such as SDS is        added, followed by addition of calcium (Ca²⁺) to complex with        the SDS, gDNA, protein, lipopolysaccharides, and other host cell        impurities, (e.g. SDS-Calcium treatment to make calcium dodecyl        sulfate) leaving the pDNA in solution. These flocculant        precipitates can then be removed by solid-liquid separation.

It is known in the art that PEG can be added to lysates to aid removalof gDNA and PEG flocculation allows debris removal with low speedcentrifugation. For example, Chen Z. Ruffner D 1998 World PatentApplication WO9816653 describes adding magnesium chloride to 1 M and PEGto 3.3% to alkaline lysates to precipitate cell debris RNA, gDNA andproteins from pDNA, to facilitate lysate clarification. In Boyd et al,Supra, 2006, PEG is used to flocculate gDNA and host cell debris from alysozyme mediated lysate. Neither of these disclosures teaches PEGflocculation of autolysates. The novel and new combination of PEGflocculation with endolysin mediated autolysis disclosed herein isdemonstrated to provide an efficient method for lysate clarification andgDNA reduction.

Low pH Extraction Process Clarification Preferred Embodiments

By way of example, consider the following methods to purify pDNA afterextraction of pDNA from endolysin or lysozyme expressing strains

-   -   1) A clarified pDNA solution may be recovered from the cell        matter in the extraction mixture by various methods known to        those skilled in the art including, but not limited to: batch        centrifugation, continuous centrifugation, flocculation of        impurities, precipitation of impurities, body feed filtration,        rotary vacuum filtration, normal flow filtration, TFF. Various        methods of clarifying lysates are discussed in Hoare et al,        Supra, 2005 and are included herein by reference.    -   2) In a preferred embodiment, the extraction mixture, or the        plasmid containing supernatant from the extraction, is subjected        to heat treatment (65-90° C.) for precipitation of proteins and        flocculation of the extraction mixture. Heat treatment may be        performed in batch or continuous mode. A preferred heat        treatment is performed in continuous flow mode in the        temperature range of 65-90° C. for 20-90 seconds (sec).    -   3) In a preferred embodiment an ionic detergent such as SDS is        added, followed by addition of calcium (Ca²⁺) to complex with        the SDS, gDNA, protein, lipopolysaccharides, and other host cell        impurities, (e.g. SDS-Calcium treatment to make calcium dodecyl        sulfate) leaving the pDNA in solution. These flocculant        precipitates can then be removed by solid-liquid separation.    -   4) Another preferred embodiment is the use of expanded bed        adsorption (EBA), fluidized bed adsorption, or batch adsorption        to recover the pDNA from the extraction mixture. Unlike        traditional packed beds, EBA columns do not require a highly        clarified feed stream. EBA columns are fed from below, creating        an expanded resin bed that allows particulates to flow around        the beads to avoid clogging. EBA is optimally operated at a flow        rate such that the upward flow velocity exceeds the        sedimentation velocity of the particulates in the feed, but does        not exceed the sedimentation velocity of the resin. Expanded bed        anion exchange adsorption resins and columns are commercially        available (e.g. Streamline DEAE, Streamline Q XL; GE        Healthcare). Application of EBA chromatography to capture pDNA        from crude lysates has been reported (Ferreira G N, Cabral J M,        Prazeres D M. 2000. Bioseparation 9: 1-6; Theodossiou I,        Sondergaard M, Thomas O R. 2001. Bioseparation 10: 31-44)    -   5) Another preferred embodiment is the use of tangential flow        filtration or cross flow filtration (TFF) for separation of        plasmid from impurities. For, example, the pDNA may be recovered        from the extraction mixture by TFF with a membrane that has a        pore size that allows the pDNA, but not the cells, to permeate        (e.g. 0.2 μm or 0.45 μm). Preferred membrane materials, pore        sizes, and operating parameters can be determined by one skilled        in the art.    -   6) Optionally, the extraction mixture is subjected to TFF with a        membrane that has a pore size that retains the pDNA, but allows        smaller impurities (e.g. RNA, proteins) to be removed in the        filtrate prior to further processing. This method could be        integrated with fermentation harvest by TFF (e.g. with a 500 kD        or 750 kD membrane) by following cell concentration with        diafiltration with the extraction solution.    -   7) Another preferred embodiment is the use of TFF for combined        cell concentration, buffer exchange, and pDNA extraction and        recovery after fermentation. For example, a membrane with a pore        size that retains cells, but allows pDNA to permeate, may be        used first as a harvest step to concentrate a fermentation        culture. Once the cells have been sufficiently concentrated to        remove the bulk of the culture medium, the plasmid extraction        solution may be added (either batch-wise or continuously by        diafiltration); continued TFF then results in recovery of the        extracted pDNA in the filtrate.        Plasmid Purification Preferred Embodiments

The resulting pDNA liquor after primary clarification may be purified ina downstream process. Boyd et al, Supra, 2006 summarizes a number ofdownstream processes that are compatible with clarified lysates; thesemethods are included herein by reference.

We contemplate utilizing host strains expressing endolysin, andoptionally plasmid-safe nucleases, to produce plasmid enriched feedstreams from high yield fermentation culture in exemplary plasmidpurification processes. The combination of endolysin mediated autolysiswith flocculation, and/or endolysin mediated plasmid extractionprocesses with exemplary downstream purification processes provide costeffective methodologies for plasmid production for gene therapy and DNAvaccination applications.

Endolysin Expression Preferred Embodiments

Expression of the endolysin and/or nuclease genes may be driven byconstitutive or, more preferably, inducible promoters. Induciblepromoters that are preferred include, but are not limited to, lambda PRand PL, other phage promoters such as T5 or T7, synthetic promoters suchas tac and trc, endogenous promoters such as lac, cold shock promoters(cspA), araBAD, stationary phase or starvation promoters, growth rate(rmf), pH (cadA), or anoxia responsive (nar) promoters. Induction can beby increased temperature (PL, tac) with thermolabile repressors (lambdarepressor, lac repressor, respectively), decreasing temperature (cspA;cold shock promoter), inducers (IPTG for tac, trc and lac; Arabinose forAraBAD) or cellular or environmental changes (e.g. entry into stationaryphase, pH or oxygen shift, glucose or amino acid starvation; reviewedin: Makrides S C. 1996 Microbiol. Rev. 60: 512-538). Alternatively, thegene may be induced by a regulated antisense RNA or by a chemicalinducer binding to a riboswitch control region in the RNA leader.

Host Strains for Autolytic Gene Expression

In a preferred embodiment, the endolysin gene is expressed from aplasmid that is compatible with a expression plasmid containing a targetprotein gen. In another preferred embodiment, the endolysin gene isintegrated into the genome. For applications where a plasmid DNA ratherthan a protein is the product, the endolysin gene is preferablyintegrated into the genome. Strain engineering can be performed in anystrain of bacteria that is suitable for plasmid production.

Compatibility of endolysin cell lines with fed-batch processes in animalproduct free NTC3019 fermentation media optimized for plasmid production(disclosed in Carnes and Williams, Supra, 2006 and included herein byreference) is demonstrated herein. Other plasmid fermentation processesknown in the art (see Background of the Invention) are also contemplatedfor use with the endolysin cell line embodiments of the invention. Aninvestigator skilled in the art could modify or create de novo, ifnecessary, endolyin expressing cell lines with alternative endolysinexpression control. For example, in alternative preferred hosts asneeded for adaptation to different plasmid fermentation processes, suchas the defined media fed-batch process of Huber, H., Weigl, G.,Buchinger, W 2005 World Patent Application WO2005097990 in the preferredhost strain E. coli JM108 (Huber, H., Pacher, C., Necina, R., Kollmann,F., Reinisch, C 2005 World Patent Application WO2005098002).

EXAMPLES

The methods and compositions of the invention are further illustrated inthe following examples. These are provided by way of illustration andare not intended in any way to limit the scope of the invention.

Example 1 Fermentation Process Optimization

Carnes and Williams, Supra, 2006 disclosed methods for fed-batchfermentation, in which plasmid-containing E. coli cells are grown at areduced temperature during part of the fed-batch phase, during whichgrowth rate is restricted, followed by a temperature up-shift andcontinued growth at elevated temperature in order to accumulate plasmid;the temperature shift at restricted growth rate improves yield andpurity of plasmid (FIG. 4).

This process takes advantage of the temperature sensitivity of high copynumber plasmids. In the preferred process, the initial temperaturesetpoint is 30° C., at which the plasmid is maintained stably at lowlevels while biomass can accumulate efficiently. During this period, thespecific growth rate is controlled at approximately μ=0.12 h⁻¹ by anexponential feeding strategy. Induction of plasmid accumulation isperformed when the cell density is in the range of 25-60 OD₆₀₀ byshifting the temperature to 42° C. and continued exponential nutrientfeeding for up to 15 hours.

After induction of plasmid production at 42° C., the cells areharvested. Typically, the fermentor is cooled directly to around 15° C.prior to harvest. It was unexpectedly found that a hold at 25° C. postplasmid production prior to cooling to around 15° C. for cell harvestimproved plasmid yield (Table 1) and quality since the percentage ofnicked plasmid was reduced after the hold (FIG. 5; sample 41 is after a2 hr 25° C. hold). Improved yields are seen when the hold is performedat 30° C. rather than 25° C. and from 0.5 hr to >3 hr holds at 25° C.(Table 1). Analysis of total cellular DNA (plasmid and genomic) frombefore and after the hold did not reveal the loss of any specificplasmid isoforms during the hold. Although the basis for the yieldimprovement is unknown, without restricting the application of theembodiments of the invention the inventors speculate that the improvedyield is the result of completion of replication of plasmid replicationintermediates, and the improved quality due to providing an opportunityfor DNA polymerase I and DNA ligase to remove replication primers andseal and supercoil the plasmid. Holding cells prior to harvest at atemperature that does not support extensive plasmid replication whileallowing plasmid repair improves yields and quality. It is likely, dueto reduced enzyme activity at low temperature, that longer hold periodswill be required at reduced temperatures, such as 15° C., than areneeded at 25° C. for maximal yield improvement.

Note that this example also demonstrates differences in yield fromdifferent plasmid backbones, with the pNTCUltra vectors surprisinglyyielding approximately twice the production yield of the gWiz derivedplasmid (Genlantis, San Diego Calif.). Although the basis for the yieldimprovement is unknown, without restricting the application of theinvention embodiments, we speculate that one possibility is that theimproved yield is the result of increased leading strand primertranscription. As a result, the pNTCUltra backbone may have maximizedRNAII promoter dependent replication priming using by using the pUCorigin in an optimal orientation relative to other DNA vaccine plasmidcomponents.

TABLE 1 Process hold step post 42° C. increases plasmid yield SpecificSpecific Specific yield Yield Yield increase Induction Induction Post T= E T = E + hold (hold) Run T = 0 Induction end T = E induction (mg/L/(mg/L/ (mg/L/ ID# Host Plasmid+ OD₆₀₀ Time OD₆₀₀ hold OD₆₀₀) OD₆₀₀)OD₆₀₀) RF41 DH5α pNTCUltra1 32 15 80 (lysis†) 2 hrs 25° C.  15.7 16.30.6 RF36 DH5α pNTCUltra1 40 11 93 1 hr 25° C. 13.9 16.2 2.3 RF48 DH5αpNTCUltra1 40 14 98 1 hr 25° C. 14.7 16.9 2.2 RF73 DH5α pNTCUltra1 4912.5 104 1 hr 25° C. 16.0 20.4 4.4 RF96 DH5α pNTCUltra2 44 12 102 1 hr25° C. 15.5 21.2 4.7 RF47 DH5α gWiz-D 52 8 86 1 hr 25° C. 8.4 9.6 1.2RF6A DH5α gWiz-D 39 8.5 85 1 hr 10° C. 8.7 8.4 −0.3 LS- DH5α gWiz-D 54 797 0.5 hr 10° C.   8.6 8.5 −0.1 01016 RF84 DH5α pNTCUltra1 38 12 95 1 hr25° C. 12.8 16.7 3.9 +++ SpoT− RF8 DH5α pNTCUltra1 38 12 78 1 hr 25° C.15.6 18.8 3.2 +++ TopA− RF67 DH5α + pNTCUltra1 31 14 76 1 hr 30° C. 14.916.3 1.4 +++ zwf RF33 DH5α pNTCUltra3 41 11.5 87 3 hrs 25° C.  14.0 17.53.5 +All plasmids contain pUC origin. pNTCUltra1-3 have differentbackbones and antigen genes. gWiz-D = gWiz derived. †Loss of ≧5 OD₆₀₀from the previous time point. +++ DH5α host strains derivatized with aSpoT mutation (decreases stringent response), TopA mutation (removes DNAendonucleaase) or overexpressing zwf gene products, respectively.

Example 2 Seed Stock Process Optimization

Creation of seed stocks from transformations performed at 30° C., ratherthan 37° C., unexpectedly increased plasmid yield, productivity and seedstock viability.

Transformations were performed using either Z competent cells (ZymoResearch, Orange Calif.) or electrocompetent cells using standardmethodologies. Briefly, electrocompetent cells (in 10% glycerol) wereelectroporated with pDNA [one of three kanamycin resistant pNTCUltra DNAVaccine plasmids containing influenza hemagglutinin (HA) genes from H1,H3 or H5 serotypes] using the BioRad (Hercules, Calif.) micropulserelectroporator per manufacturers instructions (2.5 kV, 25 uF, 100Ω, 0.1cm cuvette, 25 μL cells) and cells transferred to 0.5 mL SOC media in afalcon tube. Transformed cells were outgrown by shaking 1 hr, and thenplated onto Luria-Bertani (LB)+kanamycin plates. For Z competent cells,competent cells were made and transformed according to the manufacturersinstructions (Zymo Research). LB media was added to the transformedcompetent cells after ½ hr incubation on ice and the cells incubated for1 hr outgrowth in a shaker incubator. Incubations (post transformationshaking and post plating incubation) were performed at either 30° C. or37° C. Seed stocks were created from saturated cultures grown in LBmedia at either 30° C. or 37° C.

Shake flask and fermentation plasmid yields (performed as described inExample 1) for seed banks of the pNTCUltra influenza DNA vaccineplasmids manufactured at either 30° C. or 37° C. are summarized in Table2. The minipreps at 30° C. and 37° C. were grown from the seed stockimmediately after manufacture. Glycerol stock viability was determinedafter >1 week at −80° C. storage. The high yielding H5 construct isinsensitive to temperature, while plasmids with the H1 and H3 insertshave higher yields and seed stock viability after creation ofcryopreserved seed stocks at 30° C. rather than 37° C. No yielddifferences were observed between seed stocks made with Z competent orelectrocompetent cells.

TABLE 2 Seed stock preparation Specific Specific yield yield pNTCUltrain 30° C. in 37° C. Glycerol Plasmid Seed stock miniprep miniprep stockFermentation antigen preparation (mg/L/OD₆₀₀) (mg/L/OD₆₀₀) viable yieldH1 30° C. 1.4 1.3 Yes 252 mg/L influenza 37° C. 2.0 2.5 No Inviable cellline H3 30° C. 1.27 3.7 Yes 570 mg/L influenza 37° C. 1.59 0.67 NoInviable cell line H5 30° C. 1.5 9.8 Yes 1,290 mg/L influenza 37° C. 1.710.4 Yes 1,260 mg/L

The surprising observation of increased plasmid yield and viability ofseed stocks transformed and manufactured at low temperature is nottaught in the art. Application of this embodiment of the inventionsolved the problem of obtaining manufacturing cell lines for “difficultplasmids’ and resulted in successful production of 3/3 plasmids comparedto only ⅓ using standard transformation at 37° C. as taught in the art.

Several cell lines have been developed to improve stability ofinherently unstable plasmids through reduction in plasmid copy number.For example, the ABLE C and ABLE K strains (Stratagene, La Jolla Calif.)utilize E. coli DNA polymerase I mutation while the GBE180 cell line(DH5α-pcnB) utilizes the E. coli pcnB mutation to lower pMB1 or ColE1origin plasmid copy number (Pierson V L, Barcak G J. 1999, Focus21:18-19). The copycutter EPI400 cell line (Epicentre Technologies,Madison Wis.) contains an inducible pcnB mutation to lower pMB1 or ColE1origin plasmid copy number during transformation and propagation, andoptionally inducing copy number via induction of pcnB expression(Haskins D. 2004 Epicentre Forum 11: 5-6). However, the authors teachthe use of these cells lines for production of inherently unstableplasmids, not stable plasmids. As well, these cell lines are not optimalfor plasmid production, since the alteration of the cell line eitherpermanently reduces copy number (ABLE and DH5α-pcnB) or requires a copynumber inducer (EPI400).

It is known in the art that reducing copy number will help stability andimprove yield of expression plasmids that produce a toxic protein. Forexample, Hershberger C L, Rosteck Jr P R. 1992 European Patent EP0493926discloses various strain or plasmid modification methods to lower thecopy number of an expression vector plasmid, thereby stabilizing theexpression vector to improve production yield of the encodedheterologous polypeptide. In a recent review, Saida F, Uzan M, Odaert B,Bontems F. 2006, Current Protein and Peptide Science 7: 47-56 summarizevarious approaches to reduce plasmid copy number to improve expressionof toxic genes in E. coli. These authors do not contemplate usingreduced temperature to lower the copy number. Many of these strategiesrequire either strain or vector modifications that permanently reducecopy number, or alter the vector sequences. Such approaches are notacceptable for high yield production of existing DNA vaccine plasmids.As well, DNA vaccine plasmids are not designed to contain E. coliexpressed genes, so production of a toxic product should not beexpected. Therefore, a investigator skilled in the art would not applythese approaches to DNA vaccine plasmids.

Joshi A, Jeang K T, 1993. Biotechniques 14:883 teach that unstable HIV-1proviral genomes can be stabilized by transformation at 30° C. comparedto 37° C. This instability is probably due to recombination between thetwo long terminal repeats in the vectors. The authors do not teach thattransformation at 30° C. improves plasmid yield, or improves stabilityof non-repetitive plasmids. The Stbl2 and Stbl4 cell lines weredeveloped to improve stability of unstable insert (retroviral longterminal repeats or direct repeats) containing vectors. The manufacturer(Invitrogen, Carlsbad Calif.) indicates these cell lines must betransformed at 30° C. rather than 37° C. for maximum performance (TrinhT, Jessee J, Bloom F R. Hirsch V. 1994 Focus 16:78-80). The authors donot teach that transformation at 30° C. improves plasmid yield, orimprove stability of non-repetitive plasmids.

As indicated above, the DNA vaccine plasmids tested in Table 2 areeukaryotic expression vectors, and are not designed to express encodedgenes in E. coli. To confirm this, a version of the vector backbone withEGFP rather than influenza HA was created, and transformed into DH5αcells. Cultures containing the plasmids were grown at 37° C. untilsaturated, the cells pelleted and washed 2× with PBS, and clarified celllysates made by sonication of the cells in TE buffer (this buffer iscompatible with EGFP). No detectable EGFP was present in cell lysates,compared to control cell lines without the plasmid, as quantifiableusing a FLX800 microplate fluorescence reader with samples in black 96well assay plates. The assay was repeated using lysates fromelectrotransformed cells that were outgrown by shaking either 1 hr at37° C. or 2 hr at 30° C. and stored at 4° C. to allow EGFP maturationprior to lysate preparation. No detectable EGFP was detected in the celllysates. This demonstrates that the improvement in yield at 30° C.compared to 37° C. is not due to a transient or constitutive burst ofantigen expression after transformation or growth at 37° C. As well, thevectors are non-repetitive and do not contain direct repeats or longterminal repeats. Therefore, the surprising observation of improvedyield (and viability) of these non-expressed, non-repetitive plasmidsfrom seed stocks created at 30° C., rather than 37° C., is not taught inthe art described above. Other applications of these embodiments of theinvention can be determined by an investigator skilled in the art. Forexample, application of low temperature seed stock growth to seed stockcreation for defined media fermentation may eliminate the costly andtime consuming step of extensive prescreening of colonies to identifyrare high producing plasmids (Prather K L J, Edmonds M C, Herod J W.2006 Appl Microbiol Biotechnol 73:815-826; Chartrain, M., Bentley, L.K., Krulewicz, B. A., Listner, K. M., Sun, W., Lee, C. B 2005 WorldPatent Application WO2005078115).

Example 3 Endolysin Strain Engineering

DH5α strains containing integrated inducible endolysin genes wereconstructed using chromosome engineering. Methods for chromosomeengineering (insertion or deletion of genes) are well known in the art,for example, lambda red gam recombination (Murphy K C 1998 J. Bact. 180:2063-2071; Datsenko K A, Wanner B L. 2000 Proc. Natl. Acad. Sci. (USA);97:6640-6645). Alternatively, replication incompetent plasmids can besite specifically integrated into the genome at bacteriophage attachmentsites utilizing bacteriophage recombinase expressing plasmids. Plasmidkits, to allow integration at various bacteriophage attachment siteshave been developed (Haldimann and Wanner J Bacteriol. 2001: 6384-6393)and are generally available (E. coli genetic stock center, Yale N.H.).These methods are included herein by reference. For example, integrationof a gene at the bacteriophage HK022 attachment site requires cloning agene to be expressed into a modified integration plasmid such as pAH144(FIG. 6). This plasmid contains the R6K conditional replication origin(which requires engineered pir+ host cells such as BW23474 forpropagation; the R6K origin is non functional in DH5α) a multiplecloning site, a streptomycinR and spectinomycinR resistance marker(spec/strep) and the lambda attachment site.

pAH144 was modified herein to allow expression of cloned genes from thepR promoter (pR and C1857ts lambda repressor included on the plasmidsuch that gene expression is repressed at 30° C. but induced at 37-42°C.). The gene of interest was cloned under the control of this promoter.

pAH144 Heat Inducible Endolysin Vectors:

Vectors were made by standard restriction digestion mediated transfer offragments between vectors. All cloning was performed in the BW23474 cellline with selection on spec/strep. All clones were sequence verified.

pAH144-Lambda C1857ts repressor-PR PL (=pAH144-Lambda repressor)

The pND213 (Love C A, Lilley P E, Dixon N E 1996 Gene 176:49-53) nativestuffer protein expression vector was digested with BamHI/PstI and thesmaller fragment (2724, 1345) containing the phage lambda C1857ts lambdarepressor, PR and PL promoters upstream of a multiple cloning site waspurified. pAH144 (E. coli genetic stock center, Yale N.H.) was digestedwith BamHI/PstI and the linear vector (2.45 kb) purified. The twofragments were ligated and transformed into the BW23474 cell line.pAH144-Lambda repressor (FIG. 6) recombinants were selected onspec/strep at 35 ug/mL each, and confirmed by restriction digestion andsequencing with RgnB-f and tL3-r primers (Haldimann and Wanner Supra.2001).

pAH144-Lambda repressor-zwf

Insert genes were made by polymerase chain reaction (PCR) amplificationfrom gDNA, using primers that incorporated restriction sites to becompatible with the NcoI and EcoRI cloning sites in pAH144-lambdaRepressor parent vector (FIG. 6). This was accomplished using the actualrestriction enzymes, or, in cases where these sites were presentinternally in the PCR fragments, by digestion with AarI type IIS enzyme.The 5′ end of the primer contains 4-6 bases, then the AarI site, 4bases, then the 4 bp sticky end of the NcoI or EcoRI enzyme. Cleavage ofthe PCR product with AarI (Fermentas, Vilnius, Lithuania) cleaves after+4 and +8 (bottom strand) to generate a 4 bp sticky end. Methods for useof AarI in cloning are disclosed in Williams, Supra, 2006 and areincluded herein by reference.

To make the pAH144 lambda repressor zwf construct, the E. coli zwf gene(Genbank M55005, region spanning the ATG start codon to the stop codon)was PCR amplified from DH5α gDNA and digested to generate NcoI EcoRIfragment and cloned into NcoI/EcoRI compatible pAH144-Lambda Repressor(prepared as two fragments; NotI/NcoI: 2777, 619, 437 and EcoRI/NotI:2820, 1013). The 3 fragment ligation with zwf fragment and two boldedvector fragments was transformed into the BW23474 cell line and selectedon spec/strep and sequence verified (pAH144-Lambda Repressor-zwf).

(SEQ ID NO: 1)

pAH144-Lambda Repressor zwf-lambdaR=pAH144-zwf-lambdaR=pAH144-zwf-λR

For multiple cistron vectors, a polycistronic linker was included afterthe zwf gene to create the pAH144-lambda repressor-Zwf bicistroncompatible vector. To make the pAH144 lambda repressor zwf-lambdaRconstruct (FIG. 6), the lambdaR endolysin gene (Genbank J02459 bp45493-45969) was PCR amplified from purified lambda DASH II DNA(Stratagene, La Jolla Calif.) with primers that created flankingNdeI-EcoRI sites. The restriction digested fragment was cloned intoNdeUEcoRI cleaved pAH144-lambda repressor-Zwf vector and confirmed bysequencing. The bicistronic linker between zwf and lambdaR has thefollowing sequence.

(SEQ ID NO: 2)

Zwf gene-lambdaR endolysin gene linker

Zwf gene-TAActcgAGGAGATATACATATG-LambdaR endolysin gene-----EcoRI

TAA is Zwf stop codon, AGGA is second cistron RBS, ATG is lambdaR startcodon, CTCGAG is XhoI, CATATG is NdeI

pAH144-zwf-t4 gene e-lambdaR

The T4 gene e endolysin gene was cloned upstream of lambdaR endolysingene (downstream of zwf) as a 3 cistron system in pAH144. This allowsheat induction of endolysin (both lambda and T4) during production ofthe plasmid, potentially making more endolysin protein by having twoendolysin genes.

pAH144-zwf-lambdaR was digested with XhoI and NdeI and the 5750 bplinear vector fragment purified. The T4 gene e gene (Genbank AF158101.6bp 66997-66503) was PCR amplified from T4 gDNA [American Type CultureCollection (ATCC) Manassas Va.] using primers that created XhoI and NdeIsites for cloning, and ribosome binding sites before and after gene e(to allow expression of gene e from the upstream RBS, and lambdaR fromthe downstream RBS. The 0.53 kb PCR product was XhoI/NdeI digested, andcloned into the vector fragment. The ligation was transformed intoBW23474 and recombinants selected with spec/strep. The clone wasconfirmed by sequencing.

An investigator skilled in the art of cloning can make overexpressionvectors for other phage endolysins, lysozymes or autolysins using thestrategies and vectors described above.

Transformation of DH5α containing the pAH69 helper with the pAH144derivative plasmid to be integrated (i.e. pAH144-zwf-lambdaR andpAH144-zwf-T4 gene e-lambdaR) results in integration of the pAH144plasmid into the genome at the phage HK022 attachment site; recombinantswere selected with spec/strep and integration verified using PCR asdescribed in Haldimann and Wanner Supra. 2001.

Integration into other phage attachment sites, using pR or pAra modifiedintegration plasmids may be performed as described in Haldimann andWanner Supra. 2001, by modifying the relevant CRIM plasmids, asdescribed herein.

Arabinose Inducible Endolysin Cell Lines (araB-lambdaR):

For comparison purposes, a DH5α strain that contains arabinose induciblelambdaR endolysin (DH5α AraC λR) was created by genome mass transfer(copending application Williams U.S. patent application 60/931,890) ofgDNA from the XJa strain (Zymo Research, Seattle Wash.), a JM109derivative with lambda R endolysin integrated into the arabinose locus(Jia et al, Supra 2006).

Example 4 Endolysin Strain Fermentation

Plasmid DNA (pNTCUltra plasmids) production fermentations with DH5α, andintegrated endolysin expressing strains DH5α-pAH144-zwf-lambdaR orDH5α-pAH144-zwf-T4 gene e-lambdaR cell lines, were performed using theinducible fermentation process of Carnes and Williams, Supra, 2006.Endolysin activity was determined using a simple STET buffer lysisassay. Briefly, 15 OD₆₀₀ of cells were pelleted and resuspended in 1 mLof ice cold STET buffer containing 2% Triton X-100. The time for thecells to lyse was determined by observation of culture clearing and is ameasurement of relative endolysin activity. As summarized in Table 3,strongly autolytic cells were produced using a cell line with a singleintegrated copy of the pAH144 lambda repressor zwf-lambdaR plasmid.Surprisingly, endolysin expression (time for autolysis) with pAH144lambda repressor zwf-lambdaR was dramatically improved compared toarabinose inducible DH5α AraC λR. Combination of lambdaR and T4 gene e(pAH144-zwf-T4 gene e-λR) resulted in even more potent autolysis. ThepAH144 lambda repressor zwf-lambdaR cell line is non toxic, and glycerolstocks maintained in this cell line show no loss of viability orfermentation productivity after storage at −80° C. For example, RF135 isa repeat of the same cell line as RF101, after storage of the glycerolstock 9 months at −80° C. As well, no reduction of plasmid yield orintegrity, compared to the DH5α parent, was observed in fermentationproduction with this cell line.

TABLE 3 Autolysis using Endolysin expressing cell lines SpecificInduction Yield Volumetric Run Production Strain Induction ++ time(mg/L/ Yield ID # strain Description OD₆₀₀ (hours) OD₆₀₀) (mg/L)Autolysis† RF41 DH5α Control 32 (42° C.) 15 16.3 1200 RF48 40 (42° C.)14 16.9 1690 RF73 49 (42° C.) 12.5 20.4 2040 RF70 DH5α—AraC arabinose 28(42° C. 12 13.4 900 Yes (15 λR inducible plasmid) min lysis) endolysin(0.2% 64 (AraC-λR = arabinose 9 hr post 42° C.) RF90 induction) 41 (42°C. 11 11.8 1390 Weak (2 hr plasmid) lysis) 109 (AraC-λR = 11 hr post 42°C.) RF91 DH5α Heat inducible 44 (42° C. shift) 11 13.3 1608 Yes (2 minpAH144-zwf- zwf-endolysin lysis) λR cassette RF101 DH5α Heat inducible45 (42° C. shift) 10 14.6 1746 Yes (5 min pAH144-zwf- zwf-endolysinlysis) λR cassette RF135 DH5α Heat inducible 50 (42° C. shift) 10.5 18.02050 Yes (5 min pAH144-zwf- zwf-endolysin lysis) λR cassette RF94 DH5αHeat inducible 43 (42° C. shift) 11 11.0 665 Yes (0.5 pAH144-zwf-zwf-dual min lysis) T4 gene e- λR endolysin cassette †At harvest usingSTET assay. A typical non-autolytic DH5α fermention requires >8 hrs toclear in this assay. Autolysis of AraC-λR during 30° C. growthrestriction phase prior to induction was observed (13 min ysis). ++Plasmid induction at 42° C. is coincident with endolysin induction withheat inducible pAH144 host strains. For DH5α—AraC λR, plasmid inductionat 42° C. is independent of endolysin induction (arabinose addition).

Example 5 Heat Inducible Endolysin Cell Line NTC3012

For production of a wide variety of potential plasmids, it is preferablethat the host strain not have integrated plasmid replication origins inthe chromosome (e.g. R6K). The pAH144-zwf-λR plasmid was amplified byPCR to delete the R6K replication origin, ligated to form a circle, andintegrated into the phage HK022 attachment site in DH5α using the pAH69plasmid as described in Example 4 and below.

The pAH144-zwf-λR plasmid (FIG. 6) was PCR amplified to delete the R6Kreplication origin using the following primers.

(SEQ ID NO: 3)

R6KdF01: cgtgagcacctgcaactGTGTtgaactgctgatcttcagat cctctac(SEQ ID NO: 4)

R6KdR01: ctccagcacctgcttttACACaggaacacttaacggctgac atg

The 5.3 kb linear product was DpnI digested (to eliminate parentplasmid), AarI digested (to create compatible sticky ends for ligation),purified, and ligated (to make exonuclease resistant circles forintegration). The ligation was electroporated into pAH69 HK022integration plasmid containing DH5α cell line and colonies were selectedwith spec/strep. The cell line (NTC3012) was confirmed with PCR usingP1-P4 primers as described (Haldimann and Wanner Supra. 2001); theseprimer sites are retained in the amplified minicircle vector.

The correct integration was confirmed by PCR analysis of the 3 celllines [DH5α parent and two autolytic cell lines; NTC3012 PCR productcell line and DH5α pAH144-zwf-λR plasmid integration cell line (Example4)] using the primer pairs below.

R6K PCR product

(SEQ ID NO: 5)

R6KF01: GGCTTCTCAGTGCGTTACATC(SEQ ID NO: 6)

R6KR01: ctaaaccctcatggctaacgtact

These primers amplify the R6K replication origin region of the pAH144plasmid removed in the PCR product. As expected, no product was observedin the PCR integrated cell line NTC3012 or DH5α, and a 230 bp productwas observed with the DH5α pAH144-zwf-λR plasmid integrated cell line.

Flanking R6K PCR product

(SEQ ID NO: 7)

R6KF02: gtcagccgttaagtgttcctg (overlaps R6KDR01 primer)(SEQ ID NO: 8)

R6KR02: caagatccggccacgatgcg

These primers amplify the region flanking the R6K origin that is presentin both the plasmid and the PCR product. As expected, a 78 bp productwas detected in the NTC3012 cell line, a 419 bp product in the DH5αpAH144-zwf-λR plasmid integrated cell line, and no product in DH5α. Thisconfirms that the NTC3012 cell line has a specifically integrated PCRproduct without the R6K origin.

The pAH69 helper plasmid was eliminated, and master and working cellbanks of the autolysis cell line created. The cell line has beentransformed with several different pNTCultra plasmids and fermented inthe NTC inducible fermentation process of Carnes and Williams, Supra,2006. As expected, the cell line performs identically (growth rate,viability, plasmid yield and autolytic activity) to the pAH144-zwf-λRplasmid integrated cell line and the parent DH5α cell lines (for growthrate, viability, and plasmid yield). This demonstrates that deletion ofthe R6K origin does not adversely affect the autolytic activity of thecell line after integration, and that the integrated endolysin has noeffect on plasmid production compared to endolysin free DH5α.

A key advantage of the heat inducible NTC3012 cell line is that itrequires no inducer (e.g. arabinose) or process modification when usedin the NTC inducible fermentation process. An additional unexpectedadvantage of NTC3012 is that endolysin expression was better repressedin the absence of inducer (i.e. at 30° C. during the growth phase) thanin comparable arabinose controlled endolysin cell lines (DH5α-AraC λR).The art teaches that the arabinose promoter is more tightly regulatedthan the pR or pL heat inducible promoters (Guzman L M, Belin D, CarsonM J, Beckwith J. 1995. J. Bacteriol. 177: 4121-4130). However, while noendolysin activity was detected prior to heat induction with the NTC3012zwf-lambdaR cell line (>8 hrs autolysis), the DH5α. AraC λR demonstrateddetectable endolysin prior to the 42° C. heat shift in the absence ofthe arabinose inducer (13 minute autolysis immediately prior totemperature shift in RF90, Table 3). This may be due to unexpectedleakiness of the arabinose promoter under growth restricted conditions.

Example 6 Autolysis with STET Buffer

Cell paste harvested from shake flask cultures of autolytic cell line(DH5α pAH144-zwf-T4 gene e-λR; see Example 4) harboring the pNTCUltralplasmid was resuspended to a density of approximately 30 OD₆₀₀ with STETbuffer. Lysis occurred within 5 minutes of resuspension due toautolysis, and was marked by a visual clearing of the suspension andincrease in viscosity. Cell paste from a culture of a non-autolytic cellline, E. coli DH5α, was also resuspended to approximately 30 OD₆₀₀ withSTET buffer and two 5 mL aliquots were made. Two thousand five hundred(2500) units of Ready-Lyse™ Lysozyme (Epicentre) was added to one 5 mLaliquot of the non-autolytic, DH5α suspension.

All suspensions were allowed to age 30 minutes at room temperature(20-25° C.). The suspension of autolytic cells became the most visuallyclear. The suspension of DH5α with added lysozyme became somewhat moreclear and viscous. No cell lysis was observed in the DH5α suspension towhich no lysozyme had been added. FIG. 7 shows the three STET cellsuspension aliquots after aging 30 minutes at room temperature. The tubeon the left shows the 30 OD₆₀₀ STET suspension with non-autolytic DH5α.The tube in the middle shows the 30 OD₆₀₀ STET suspension withnon-autolytic DH5α to which 2500 units Ready-Lyse™ Lysozyme (Epicentre,Madison Wis.) was added. The tube on the right shows the 30 OD₆₀₀ STETsuspension of cell line DH5α pAH144-zwf-T4 gene e-λR pNTCUltral, whichlysed efficiently without addition of lysozyme. This demonstrates thatendolysin can substitute for lysozyme in a STET/lysozyme process.

Example 7 Effect of the Composition of Lysis Solution Containing Glucoseand/or Triton X-100 and EDTA

Cell paste from an lambdaR autolytic fermentation was resuspended at aconcentration of 10 mL buffer per gram (g) wet cell weight (WCW) with 50mM Tris pH 8.5 buffer containing varying amounts of Triton X-100 (0.04%or 2.0%), EDTA (1 mM or 50 mM), and glucose (0% or 8%); the fullfactorial was used, giving 8 different buffer compositions. Theresuspended cells all lysed by autolysis and were incubated at 37° C.for 35 minutes, then centrifuged at 13000 g for 10 minutes. One (1) mLof supernatant from each of the 8 lysates was heated to 68° C. for 30minutes, then centrifuged at 13000 g for 10 minutes. Equal volumesamples from each lysate were analyzed by agarose gel electrophoresis(FIG. 8). The best plasmid quality was obtained with the combination of8% glucose and 50 mM EDTA.

Example 8 Autolysis with Solution Containing PEG and Different Amountsof NaCl

Five cell paste samples of 0.3-0.5 g WCW from a lambdaR autolyticfermentation were each resuspended with a different lysis solution at 10mL per g WCW. The lysis solutions all contained 7.5% PEG-8000, 50 mMTris pH 8.0, 10 mM EDTA, and 1% Triton X-100. The difference in thelysis solutions was the concentration of NaCl, which had a range of 0M-0.4 M NaCl. Each of the 5 cell suspensions underwent autolysis and wasaged for 40 minutes at room temperature. Then, 0.5 mL samples werecentrifuged at 13000 g for 10 minutes to pellet the insoluble materialand give a clear supernatant. The supernatants were recovered from thepelleted material. The pelleted material from the autolysis containing0.4M NaCl was resuspended and extracted with 0.5 mL of TE buffer, andthen pelleted again by centrifugation; the clear supernatant wasrecovered. Samples from the clear autolysis and TE extractionsupernatants were pre-stained with SYBR Green II and analyzed by agarosegel electrophoresis (FIG. 9).

This example shows that in a lysis buffer containing approximately 7.5%PEG-8000, increasing concentrations of NaCl reduce the solubility ofDNA. FIG. 9 shows that the supercoiled pDNA is solubilized in autolysiswith the solution containing up to 0.2M NaCl. The plasmid is notsolubilized in autolysis when the solution contains 0.3M or greaterNaCl. Lanes 2, 3, and 4 in FIG. 9 show decreasing amounts of highermolecular weight DNA, such as gDNA, while the pDNA remains soluble asthe NaCl concentration increases from 0M, to 0.2 M. Thus, a selectivesolubilization of pDNA was achieved. Neither the gDNA nor the pDNA wassolubilized when the autolysis solution contained 0.3 M or 0.4 M NaCl,as shown by the absence of plasmid or gDNA bands in lanes 5, 6, and 7 ofFIG. 9. In these samples the RNA remained soluble. The supernatant fromthe autolysis that did not contain any NaCl was much more viscous thanthe autolysis supernatants that contained NaCl.

To further show that the plasmid was not solubilized in an autolysissolution containing 7.5% PEG-8000, 50 mM Tris pH 8.0, 10 mM EDTA, 1%Triton X-100, and 0.4M NaCl, the pellet of insoluble material obtainedfrom this autolysis after centrifuging was resuspended in 0.5 mL TEbuffer to dissolve the DNA. The DNA solubilized from the pellet is shownby Lane 8 in FIG. 9.

Example 9 Composition of a Lysis Solution in which pDNA is SelectivelySoluble

The following solution is useful for performing autolysis or lysozymelysis and is referred to herein as “PNL buffer”.

PEG-8000 7.5% EDTA 10 mM Tris 50 mM NaCl 0.15M pH 8

Host cell gDNA, cell debris, and other impurities are largely insolublein this solution. However, pDNA is soluble in this solution. Theinsoluble gDNA and other insoluble host cell impurities may be removedby solid-liquid separation, such as settling, centrifugation, orfiltration. Addition of small quantities of detergent or surfactant aidslysis (e.g. 0.02-5% Triton X-100 or SDS).

Example 10 Preparation of Clarified Lysate by Autolysis in PNL Bufferfor pDNA Recovery

Approximately 40 g WCW of cell paste from an autolytic E. coli hoststrain fermentation (cell line DH5α pAH144-zwf-T4 gene e-λR/pNTCUltralplasmid) had a plasmid content of approximately 122 mg pDNA. The cellpaste was thoroughly suspended with 520 mL of PNL buffer as described inExample 9: Composition of a lysis solution in which pDNA is selectivelysoluble. Approximately 1.3 mL of 20% Triton X-100 was added to thesuspension and mixed well, and then allowed to stand still at roomtemperature. Within 15 minutes, autolysis had occurred and insolubleimpurities (e.g. including gDNA and cell debris) separated from theliquid lysate and floated. This insoluble material was removed bypouring the lysate through two layers of Miracloth and filtrationthrough a 0.2 μm polyethersulfone capsule filter. Approximately 500 mLof clarified lysate containing the pDNA was obtained by this process.

Example 11 Preparation of Clarified Lysate by Autolysis in PNL Bufferwith SDS and Ca²⁺ for pDNA Recovery

Approximately 109 g WCW of cell paste (RF91) from an autolytic E. colihost strain was thoroughly suspended with 1.4 L of PNL buffer composedas described in Example 9: Composition of a lysis solution in which pDNAis selectively soluble. Throughout this autolysis process, 0.5 mLsamples were taken and centrifuged to obtain a clear lysate for agarosegel electrophoresis analysis. One (1) μL from each sample was run onagarose gel electrophoresis, and the gel was post-stained with SYBRGreen II. The cell suspension was mixed with a motor driven impeller for30 minutes to insure complete resuspension (FIG. 10, lane 1). Seven (7)mL of 20% SDS was added to the suspension and mixing continued for 60minutes at room temperature. FIG. 10, lanes 2 and 3 show the lysatesamples after 30 minutes and for the full 60 minutes, respectively, andit can be seen that pDNA release was complete after 30 minutes. Then,3.36 mL of 5M CaCl₂ was slowly added with continuous mixing (FIG. 10,lane 4). The mixture still appeared somewhat viscous, and another 0.14mL of 5M CaCl₂ was added, and a decrease in the amount of the highestmolecular weight DNA is clear in FIG. 10, lane 5, without anysignificant plasmid loss. The viscosity of this autolysis mixturedropped and mixing was stopped. The autolysis mixture was allowed tostand still at room temperature. After 50 minutes, the insolublematerial settled to the bottom of the container. The autolysis mixturewas clarified by centrifuging for 20 minutes at 12000 g, followed byfiltration through a 0.2 μm polyethersulfone capsule filter.Approximately 1.3 L of clarified lysate containing the pDNA wasrecovered.

Example 12 Preparation of Clarified Lysate by Lysozyme Lysis in PNLBuffer for pDNA Recovery

Lysozyme can be substituted for endolysin for use with PNL buffer inpreparing a clarified lysate as described below. An investigator skilledin the art would add approximately 13 volumes of PNL buffer per g WCW ofcell paste from a non-autolytic E. coli host strain such as DH5α andthoroughly suspended the cells as described in Example 9: Composition ofa lysis solution in which pDNA is selectively soluble). Recombinantlysozyme is added to a final concentration as determined by one skilledin the art. Triton X-100 is added to the suspension and mixed well, andthen allowed to stand still at room temperature. The mixture isincubated for lysis to occur and insoluble impurities (e.g. includinggDNA and cell debris) to separate from the liquid lysate. This insolublematerial is then removed by filtration to obtain a clarified lysate thatcontains the pDNA.

Example 13 Purification of pDNA from a Clarified Lysate Produced byAutolysis with PNL Buffer

Approximately 111 g WCW of cell paste was harvested from 450 mL of afermentation (RF91) with an autolytic E. coli host strain. This cellpaste had a plasmid content of approximately 724 mg pDNA. The cell pastewas thoroughly suspended with 1.4 L of PNL buffer (described in Example9: Composition of a lysis solution in which pDNA is selectivelysoluble). Seven (7) mL of 20% Triton X-100 was added to the suspensionand mixed well, and then allowed to stand still at room temperature.Autolysis occurred, and the insoluble material was separated from theliquid lysate by centrifugation and 0.2 μm filtration. Approximately 1.4L of clarified lysate containing the pDNA was recovered. Approximately2.8 L of 0.675 M NaCl was mixed with the clarified lysate, to make afinal NaCl concentration of 0.5M NaCl. Approximately 105 mL of 20%Triton X-100 was added to make a final Triton X-100 concentration ofabout 0.5%. This lysate solution was chilled in an ice water bath for 30minutes. Then the mixture was pumped through a Pall Mustang™ Q anionexchange membrane capsule with a 60 mL membrane bed volume. The anionexchange capsule was then washed with 5.3 L of 0.6M NaCl, 25 mM Tris, 1mM EDTA, pH 8.0. The plasmid was eluted from the anion exchange capsulewith 794 mL of a solution containing 1M NaCl, 25 mM Tris, 1 mM EDTA, pH8.0. The plasmid concentration of the elution pool was 0.564 mg/L andthe absorbance ratio A₂₆₀ nm/A₂₈₀ nm was 1.88. A total of 447 mg ofpurified pDNA was recovered by this process, which is a 62% yield.

Example 14 Purification of pDNA Using Autolysis and Filter Membranes

Plasmid DNA from 260 mL of clarified lysate, prepared as described inExample 10: Preparation of clarified lysate by autolysis in PNL bufferfor pDNA recovery, was purified by a non-chromatographic method.Approximately 15.6 mL of 5 M NaCl was mixed with the 260 mL of clarifiedlysate, making a final NaCl concentration of 0.42 M. The pDNA becameinsoluble and precipitated, which was observed visually by the hazyappearance of the mixture.

This precipitation mixture was slowly pumped through a Whatman Polycap36TC polyethersulfone filter capsule. The entire volume was pumpedthrough the filter capsule and the filtrate was clear (i.e. the haze wasremoved). Then, a mixture of 100 ml, of PNL buffer and 6 mL of 5 M NaCl,in which plasmid is not soluble, was pumped through the filter capsuleas a wash. Pumping was continued until the liquid in the filter capsulewas removed as much as possible. The filter capsule was then filled withTE buffer pH 8.0, in which pDNA is soluble. The TE buffer was slowlypumped through the filter capsule and the TE filtrate was collected. Twoseparate 5 mL fractions were collected first, followed by a third 75 mLfraction. Samples of the clarified lysate, filtrate after plasmidprecipitation, wash filtrate, and the three TE filtrate fractions wereanalyzed by agarose gel electrophoresis. As shown in FIG. 11 the plasmidwas precipitated by the addition of NaCl to 0.42 M to the clarified PNLbuffer lysate. This precipitated pDNA was removed by filtration, and wasthen recovered by dissolving in TE buffer as the TE buffer was pumpedthrough the filter. As shown by FIG. 11, the pDNA was present in thesecond 5 mL TE filtrate fraction and in the 75 mL TE filtrate fraction.The DNA concentration and absorbance ratio A_(260 nm)/A_(280 nm) ofthese two fractions was:

Second 5 mL fraction: 0.41 mg/mL A_(260 nm)/A_(280 nm) = 1.8 75 mLfraction: 0.36 mg/mL A_(260 nm)/A_(280 nm) = 1.8

Thus, a total of 29 mg of plasmid was recovered in thisnon-chromatographic method. RNA removal was achieved since the RNAstayed in solution and was removed in the filtrate as the precipitatedpDNA was retained on the filter membrane.

Example 15 Purification of pDNA by Autolysis and Cross Flow Filtration

Approximately 20 g WCW of autolytic cell paste (RF91) was resuspendedwith 260 mL of PNL buffer (described in Example 9: Composition of alysis solution in which pDNA is selectively soluble). After completeresuspension, Triton X-100 was added to a final concentration of 0.1%.Autolysis occurred rapidly. The lysate was centrifuged to remove theinsoluble material. The supernatant was recovered and heated to 65° C.for 20 minutes, which caused the lysate to become very turbid due to theprecipitation of proteins. The precipitated material was then removed bycentrifugation and filtration. Approximately 200 mL of clear lysate wasrecovered.

This lysate was buffer exchanged and concentrated by TFF with a 300 kDmolecular weight cut-off polyethersulfone membrane cassette with mediumscreen channels. The membrane area was 0.2 ft². A trans-membranepressure (TMP) of 15 psig was maintained. The filtrate was recirculatedback to the feed for the initial 20 minutes. Then, diafiltration with TEbuffer pH 8.0+1% Triton X-100 was started. The total diafiltrationvolume was 1000 mL. Then, the retentate plasmid pool was concentrated to100 mL, followed by diafiltration with 400 mL of TE buffer pH 8.0.Finally, the retentate plasmid pool was concentrated to a final volumeof 63 mL, which had a plasmid concentration of 1.34 mg/mL. Concentrationand buffer exchange of plasmid lysates can significantly increaseplasmid binding capacity of subsequent chromatography steps.

Example 16 Purification of pDNA by Autolysis and a Non-ChromatographicProcess

Seven (7) g WCW of autolytic cell paste was resuspended with 200 mL PNLbuffer plus 1% Triton X-100 (described in Example 9: Composition of alysis solution in which pDNA is selectively soluble). Autolysisoccurred, and then the lysate was centrifuged to remove the insolublematerial. The lysate was then heated to 65° C. for 30 minutes, whichcaused the lysate to become very turbid due to protein precipitation.The precipitated material was removed by centrifugation, and the clearlysate was pooled into a clean bottle. Then, 10 mL of 5 M NaCl was mixedwith the lysate to precipitate the pDNA, and this was chilled on ice for10 minutes. The precipitated pDNA was pelleted by centrifugation at12000 g for 20 minutes. The supernatant contained the soluble RNA andwas removed. The pDNA was dissolved in 200 mL of 50 mM Tris pH 8.0.Hydrated calcium silicate was added to the pDNA solution to a finalconcentration of 20 g/L. This mixture was kept suspended by rocking forabout 13 hours at room temperature. The hydrated calcium silicate wasthen removed by 0.45μ. filtration. The pDNA was precipitated by additionof 24 mL 3M sodium acetate pH 5.2 and 171 mL of 2-propanol. Theprecipitation was chilled for 10 minutes and then centrifuged for 30minutes at 12000 g to pellet the pDNA. The DNA pellet was washed with70% ethanol and then air dried for 5 minutes. Finally, the pDNA wasdissolved with 10 mL of TE buffer pH 8.0. This pDNA solution had aplasmid concentration of 0.58 mg/mL and an absorbance ratioA_(260 nm)/A_(280 nm) of 1.89. The plasmid yield from this process wasapproximately 48%. Samples from this process were analyzed by agarosegel electrophoresis (FIG. 12).

Example 17 PEG Autolysis Process with Selective Precipitations for gDNA,Protein, and RNA Removal

-   -   1. Resuspend with PEG-NaCl Lysis buffer (PNL Buffer)        -   50 mM Tris        -   10 mM EDTA        -   7.5% PEG8000        -   0.15M NaCl        -   TritonX-100        -   pH 8.5    -   2. After autolysis remove insoluble material which contains host        gDNA, cell debris, and other host cell impurities.    -   3. Heat lysate to 65° C. to 75° C. to precipitate protein,        remove precipitated material.    -   4. Add sufficient NaCl to cause precipitation of pDNA while        leaving RNA in solution, preferably to a final concentration        greater than 0.3M, more preferably to about 0.4M NaCl. Recover        precipitated plasmid.

Example 18 Extraction of Plasmid from Fermentation Cells; Evaluation ofMethods Described in the Art

Cells from a non-autolytic pDNA fermentation were utilized withoutfreeze thaw to evaluate the chemical extraction methods of Baker andTaylor, Supra, 2003. Extraction was performed as described in Baker andTaylor, Supra, 2003. Five (5) OD-units of cells were resuspended to 100OD₆₀₀ in either: 1) water; 2) 10 mM Tris 1 mM EDTA, pH 8.0 (TE buffer);3) 10 mM Tris, 10 mM EDTA, pH 8.0; or 4) 1% Triton X-100. All extractswere incubated at 37° C. for 10 min. No plasmid release was observedunder any of these conditions, while RNA and gDNA were released. RNA wasreleased to a higher level with 10 mM Tris, 10 mM EDTA pH 8.0, but noRNA was released with Triton X-100. This demonstrates that the art(Baker and Taylor, Supra, 2003) which teaches how to extract plasmidfrom shake flask cells with low ionic strength buffers is not effectivewith plasmid fermentation cells.

Cells from a second non-autolytic pDNA fermentation were utilized with asingle freeze thaw to further evaluate chemical extraction methods ofBaker and Taylor, Supra, 2003 and to test the conditions for nucleicacid extraction described by Clark and Kacian, Supra, 1998. Inducedcells were from fermentation cells after plasmid induction (i.e. aftergrowth at 42° C.) and were 78 OD₆₀₀/mL, 741 mg plasmid/L (9.5 mgplasmid/OD₆₀₀/L). Uninduced cells were from fermentation cells at 30° C.before temperature shift and were 55 OD₆₀₀/mL, 93.5 mg plasmid/L, 1.7 mgplasmid/OD₆₀₀/mL. Extractions were performed on 5 OD₆₀₀ of cellsresuspended to 50 OD₆₀₀/mL with: 1) water; 2) TE; 3) 7.4 mM HEPES 7.4,0.7% Triton X-100, 10 mM EDTA; 4) 7.4 mM HEPES 7.4, 0.7% Triton X-100,10 mM EDTA+1% SDS; 5) 10 mM Tris, 10 mM EDTA, pH 9.3. Extracts wereincubated at 37° C., 65° C., or 80° C. All SDS containing samples lysedunder these conditions. No significant plasmid release was observed inunlysed samples. gDNA was released at 65° C. and 80° C. treatment. Withuninduced cells, gDNA was release with water, TE and Triton X-100/EDTAat 65° C. and 80° C., but not at 37° C. With induced cells, only TritonX-100/EDTA at 65° C. and 80° C. released gDNA. Coomassie stainedSDS-PAGE gels of 80° C. samples reveals protein release with water, TEand Triton/EDTA with uninduced samples, and only Triton X-100/EDTAreleased protein with induced samples.

This demonstrates fermentation grown cells are generally resistant toDNA and protein extraction. This also demonstrates that the art whichteaches nonlytic DNA extraction with 7.4 mM Hepes, pH 7.4, 0.7% TritonX-100, 10 mM EDTA (Clark and Kacian, Supra, 1998) does not extract pDNAfrom plasmid fermentation cells, while the chemical extraction methodsof Baker and Taylor, Supra, 2003 also do not release plasmid fromplasmid fermentation cells.

Example 19 Non Lytic Chemical Extraction of Plasmid

Non lytic chemical extraction of proteins has been reported, using avariety of solutions (Choe W S, and Middelberg A P J. 2001 BiotechnolBioeng. 75: 451-455). The feasibility of such extraction methods forplasmid release has not been evaluated.

Extraction of DNA from uninduced high copy fermentation cells (Example18) was performed using Novagen bugbuster reagent (Novagen, MadisonWis.) and Pierce B-per reagent (Pierce Rockford Ill.). No lysis wasobserved after 30 min. After rotation overnight at room temperature,some viscosity increase was observed in both samples. Despite lysis,very little plasmid was extracted.

Extraction of DNA from induced high copy fermentation cells (Example 18)was performed using: 1) 6 M urea 1 mM EDTA pH 3; 2) 4 M urea, 10%sucrose, 1 mM EDTA, 50 mM glycine, pH 9.5; 3) Pellet from 1, resuspendedin 8 M urea, 3 mM EDTA; 4) Pellet from 2, resuspended in 0.5% aceticacid, 1% NP40. Uninduced cells were extracted with: 5) 100 mM glycine,pH 3; 6) 50 mM MOPS, 200 mM MgCl₂, pH 3. All extractions were for 10 minat room temperature. The suspensions were clarified by centrifugation,Phenol/Chloroform extracted, supernatants precipitated with ethanol,pellets resuspended in TE and DNA and RNA resolved on agarose gels.Nucleic acids were visualized by poststaining with SYBR Green II.Limited plasmid and gDNA extraction was observed with conditions 1 and2, less extraction with 3 and 6, and no extraction with 4 and 5. Plasmidyield and extraction quality were very low in all conditions.

Urea/EDTA extraction was further evaluated. Induced high copyfermentation cells were washed with PBS and resuspended to 6.5 OD₆₀₀/mLin 8M urea, 3 mM EDTA, 0.1 M Tris, pH 9.2 (Choe and Middelberg, Supra,2001). Nucleic acids were extracted in presence of: 1) no additionaladditive; 2) 0.25% final Cetyl trimethylammonium bromide (CTAB); 3) 0.1M CaCl₂; 4) 3% PEG 3350; 5) 35 mM spermidine. Samples were rotatedovernight at room temperature. Flocculation was observed in sample 2,sample 3 was granular, sample 5 slightly clumpy, samples 1 and 4 werehomogeneous. No lysis was observed. The samples were centrifuged (largepellet was observed in sample 2), the supernatants extracted withPhenol/chloroform to remove protein, and samples resolved on an agarosegel. Nucleic acids were visualized by SYBR Green II poststain. Plasmidand gDNA was present in the PBS wash and urea extractions with andwithout PEG. CTAB and CaCl₂ prevented DNA extraction. Plasmidextraction, with less gDNA contamination, was observed in the presenceof spermidine. Overall, extracted plasmid yields were low and thereleased plasmid was heavily contaminated with gDNA.

Extractions were performed on uninduced and induced cells with 8M urea,3 mM EDTA, 0.1 M Tris, pH 9.2 solution for 1 hr (rather than overnight).Lysis was observed with uninduced cells, and the sample became highlyviscous. In induced cells, plasmid extraction was observed after 1 hr,with high amounts of gDNA, but not RNA contamination. The releasedplasmid was intact but yields and purity were low.

Collectively, these results demonstrate that while analytical amounts ofplasmid can be extracted from fermentation cells using a variety ofchemical treatments, the yield and purity are low. A method to improvethe quality and yield of plasmid extraction is needed.

Example 20 Extraction of Plasmid from Endolysin Containing Cells

Various plasmid extraction methods outlined in Examples 19 and 20 wereevaluated with endolysin containing autolytic cells. Surprisingly,plasmid was efficiently extracted from endolysin cells using a varietyof conditions, including:

H₂O,

1 mM EDTA,

0.1% Triton X-100,

10 mM Tris, (pH 8.0),

50 mM Tris, (pH 8.0),

50 mM Tris, (pH 8.0), 1 mM EDTA,

50 mM Tris, (pH 8.0) 10 mM EDTA

and sucrose containing buffers.

Plasmid extraction was efficient with freshly harvested fermentationcells or frozen cell pellets. This demonstrates that the failure toextract plasmid from fermentation cells utilizing methods described inthe art is due to the barrier function of the cell wall, and can beovercome by inclusion of endolysin in the cell cytoplasm.

Example 21 Extraction of pDNA from Endolysin Containing Cells UsingAcetate Solutions

In order to manufacture pDNA at large scale, the extraction processideally should not result in a very viscous mixture. It was surprisinglydetermined that extraction at acidic pH prevents the mixture frombecoming very viscous. Acetate solutions are preferred as buffers in thedesired pH range. To evaluate the effect of acetate salt concentrationon extraction and mixture properties, 2 g aliquots of frozen autolyticNTC3012 cell paste containing 5.2 mg plasmid per g WCW were resuspendedwith 20 mL (10 volumes) of solution containing 10 mM EDTA and 0-0.4 Mpotassium acetate or 0-0.4 M sodium acetate, pH 4.8-4.9; 1 mL aliquotsfrom these cell suspensions were additionally treated with 0.1% TritonX-100 (B samples) or 1% PEG8000 (C samples) (Table 4).

TABLE 4 Extraction of pDNA from endolysin containing cells using acetatesolutions A B C 1 10 mM EDTA 1A + 0.1% Triton X-100 1A + 1% PEG8000 20.1M potassium acetate, 2A + 0.1% Triton X-100 2A + 1% 10 mM EDTAPEG8000 3 0.2M potassium acetate, 3A + 0.1% Triton X-100 3A + 1% 10 mMEDTA PEG8000 4 0.3M potassium acetate, 4A + 0.1% Triton X-100 4A + 1% 10mM EDTA PEG8000 5 0.4M potassium acetate, 5A + 0.1% Triton X-100 5A + 1%10 mM EDTA PEG8000 6 10 mM EDTA 6A + 0.1% Triton X-100 6A + 1% PEG8000 70.1M sodium acetate, 7A + 0.1% Triton X-100 7A + 1% 10 mM EDTA PEG8000 80.2M sodium acetate, 8A + 0.1% Triton X-100 8A + 1% 10 mM EDTA PEG8000 90.3M sodium acetate, 9A + 0.1% Triton X-100 9A + 1% 10 mM EDTA PEG800010 0.4M sodium acetate, 10A + 0.1% Triton X-100 10A + 1% 10 mM EDTAPEG8000

All extraction mixtures containing potassium acetate (2-5) or sodiumacetate (7-10) were non-viscous. Extractions 1 and 6, which contained noacetate salt, were viscous.

Samples from the A group in Table 4 were centrifuged immediatelyfollowing resuspension and the supernatant was saved. Then theextraction mixtures were held at room temperature for 2 hours, afterwhich samples were centrifuged to obtain clear supernatant. One (1) μLof each extraction supernatant was run on 1% agarose (FIG. 13). As shownby the gel picture in FIG. 13, plasmid extraction was rapid and did notappear to increase after 2 hours of incubation. Sodium acetate resultedin the best plasmid extraction. The addition of Triton X-100significantly increased the extraction of plasmid. The highest amount ofplasmid was extracted using 0.4 M sodium acetate, 10 mM EDTA, with 0.1%Triton X-100.

Example 22 Effects of pH and Sodium Acetate Concentration

To further evaluate the effect of sodium acetate concentration and pH onplasmid extraction and viscosity, samples of the same frozen cell pasteused in Examples 21 were resuspended with 10 volumes of 0.4-1.0 M sodiumacetate, 10 mM EDTA, at pH 4.5, 5.0, and 5.5. Triton X-100 was added to0.1% after resuspension. The pH 4.5 extractions were all non-viscous. AtpH 5.0 the extractions were more viscous, and the viscosity increasedwith increasing sodium acetate concentration. The extractions at pH 5.5were most viscous. Plasmid release was maximal at pH 5.0 and pH 5.5(FIG. 14). This example, along with Example 21, shows that plasmid canbe efficiently extracted over a wide range of acetate saltconcentrations at a slightly acidic pH.

Example 23 Large Scale Plasmid Isolation by Extraction of pDNA fromEndolysin Containing Cells

Approximately 100 g WCW of frozen cell paste of endolysin containingcells from a fermentation with a specific plasmid yield of approximately3.1 mg pDNA/g WCW was resuspended with 1000 mL of 0.4 M sodium acetate,10 mM EDTA, pH 4.8 bp vigorous mixing on a stir plate. Triton X-100 wasadded to a final concentration of 0.1% w/v, and mixing was continued tothoroughly distribute the Triton X-100. The mixture was incubated at 37°C. for 20 minutes. The mixture remains non-viscous and was centrifugedfor 25 minutes at 12000 g to remove cells and debris. Approximately 850mL of supernatant was recovered with a total DNA concentration of 0.32mg DNA/mL, 96% as pDNA and 4% as gDNA. Thus, the plasmid yield from theextraction was 261 mg pDNA (an 84% step yield).

Example 24 Kit for Extraction of Plasmid from Cells

The requirement for endolysin in the low pH plasmid extraction processof Examples 21-23 was further evaluated as follows. Plasmid wasextracted from inducible process fermentation cells from 4 independentcell lines. 1) Plasmid 1, 5.2 kb NTC8382-41H-HA antibiotic freebackbone, NTC3016 autolytic cell line [NTC8382-41H-HA is a influenza DNAvaccine plasmid and NTC3016 is a autolytic cell line derived fromNTC3012 (Example 5) described in copending application Williams JA, USPatent Application U.S. 60/932,160], 2) Plasmid 2, 6.5 kb kanamycinresistant (kanR) pUC origin backbone, DH5α cell line (no endolysin), 3)Plasmid 2, pAH144-zwf-lambdaR integrated cell line (Example 4) 4)Plasmid 3, 22 kb ampicillin resistant (ampR) backbone,pAH144-zwf-lambdaR integrated cell line.

Extraction was performed as follows. Twenty (20) g frozen cell paste wasresuspended in 10 volumes (200 mLs) of low pH extraction buffer (30 mMsodium acetate, 50 mM EDTA, 8% sucrose, pH 4.8).

Sample A was removed. Triton X100 was added to 0.1%, and Sample Bremoved after 10 minutes at ambient temperature, and Sample C removedafter an additional 20 minutes incubation at 37° C. (typically the cellsreach 30° C. in this time). Each sample (1 mL) was clarified bycentrifugation, and 1 μL of each supernatant resolved on an agarose gel.Sample D was post centrifugation of the entire 200 mL extraction.

The results are shown in FIG. 15 and demonstrate that endolysin wascritical for extraction (no extraction with DH5α cells). The resultsalso demonstrated that low pH extraction releases essentially 100% of 3different plasmids, and was not limited based on size (plasmid 3 is 22kb) or backbone (antibiotic free versus kanR or ampR). Variable amountsof plasmid were extracted without Triton X-100.

A followup low pH extraction of inducible fermentation cells fromPlasmid 2, 6.5 kb kanR pUC origin backbone, DH5α cell line (noendolysin), was performed, to determine if lysozyme can substitute forendolysin. A control extraction of the same plasmid from thepAH144-zwf-lambdaR integrated cell line (Example 4) was also performed.Approximately 20 g frozen cell paste was resuspended in 10 volumes (200mL) of low pH extraction buffer (30 mM sodium acetate, 50 mM EDTA, 8%sucrose, pH 4.8). The pH was adjusted to 5.2 with 0.2 M NaOH. Sample Awas removed. Three 50 mL samples were removed and treated as follows:Extraction 1; HEWL (Sigma, St Louis Mo.) added to 5000 U/mL. Extraction2; Ready-Lyse lysozyme (Epicentre, Madison Wis.) added to 1200 U/mL.Extraction 3; Negative control (no lysozyme added). Triton X-100 wasadded to 0.1%, and the suspension incubated 40 minutes at 37° C. SampleB was then removed from each extraction. (Samples B1, 2, and 3respectively: for the pAH144-zwf-lambdaR integrated cell line, only B3was performed). Each sample (0.5 mL) was clarified by centrifugation,and 1 μl of each supernatant resolved on an agarose gel. The results areshown in FIG. 16, and demonstrate that either lysozyme can substitutefor endolysin in the low pH extraction process. This is surprising,since HEWL has low activity at this pH range (Jensen and Kleppe, Supra,1972).

The surprising observation that lysozyme can substitute for endolysin inlow pH plasmid extraction enables creation of kits for plasmidextraction from general laboratory strains of E. coli. While manypossible kits can be envisioned, one possible low pH plasmid extractionkit is demonstrated below, linking ‘autolyte’ extraction buffer with theQiaprep miniprep spin column kit (Qiagen, Germany).

-   -   1. Centrifuge amount of cells expected to contain 10-20 μg of        plasmid 1 min in microcentrifuge    -   2. Resuspend cells in 100 μL autolyte resuspension buffer. †    -   3. Incubate 5-10 min room temperature.    -   4. Pellet autolyte extracted cells by centrifuging 0.5-1 mL at        about 12000 g for 2-5 min (in a bench top centrifuge).    -   5. Remove supernatant to fresh tube by pipetting.    -   6. Add 5 volumes Qiagen Buffer PB and mix.    -   7. Apply to the QIAprep spin column.    -   8. Centrifuge for 60 s. Discard the flow-through.    -   9. Wash QIAprep spin column by adding 0.5 mL Buffer PB and        centrifuging for 1 min.    -   10. Discard the flow through, and wash QIAprep spin column by        adding 0.75 mL Buffer PE and centrifuging for 1 min.    -   11. Discard the flow-through, and centrifuge for an additional 1        min to remove residual wash buffer.    -   12. Place the QIAprep column in a clean 1.5 mL microcentrifuge        tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris, pH 8.5) to        the center of each QIAprep spin column, let stand for 1 min, and        centrifuge for 1 min.    -   13. Optionally add another 50 μL Buffer EB to the center of each        QIAprep spin column, let stand for 1 min, and centrifuge for 1        min. Performing the elution twice with 50 μL Buffer EB insures        complete recovery of pDNA.        († e.g. 30 mM sodium acetate, 50 mM EDTA, 8% sucrose, 0.1%        Triton X-100, 5000 units/mL lysozyme, 100 μg/mL RNase, pH 5.2;        acceptable modifications include 30-400 mM sodium acetate, 0-50        mM EDTA 0-100 μg/mL PEI, pH 4.7-5.5.)

This kit eliminates several steps required for alkaline lysis (P1, P2,N3) resulting in superior speed and flexibility. The purified plasmid isof a high quality, with excellent 260/280 ratio and integrity by gel andlow levels of gDNA (due to retention of gDNA in the cell pellets). Aninvestigator skilled in the art can adapt the plasmid extraction methodembodiments of the invention to vacuum manifold devices, other spincolumn devices or other plasmid purification components of commerciallyavailable kits.

Example 25 Extraction of Protein from Cells

Low pH extraction was evaluated for utility in protein purification. TheNTC3012 endolysin expressing cell line (Example 5) was transformed withpVEX-EGFP and pVEX-AmCyan (pVEX is a standard ampR IPTG induciblebacterial expression plasmid). These plasmids express the indicatedfluorescent proteins in the cytoplasm of E. coli after IPTG induction.Fifty (50) mL bacterial cultures were grown in LB media containingampicillin, and EGFP or AmCyan expression induced by addition of IPTGand subsequent 4 hrs growth at 42° C. (to coinduce endolysin). Five (5)mL samples were resuspended to 5 OD₆₀₀/mL in autolyte buffer (30 mMsodium acetate, 8% sucrose, 50 mM EDTA, 0.1% Triton X-100, pH 5.2) andeither extracted (10 min at 37° C.) or sonicated (positive control). Thesuspensions were centrifuged 2 min in a microcentrifuge. The AmCyanextracted pellet was resuspended in the original volume of 50 mM sodiumphosphate 0.3 M NaCl, pH 7.2, and the cells extracted 5 minutes ambienttemperature, then repelleted. Release of EGFP or AmCyan in thesupernatants was quantified using the FLX800 microplate fluorescencereader with black 96 well assay plates. Integrity was assessed byCoomassie stained SDS-PAGE gel analysis as described in Williams andHodgson, Supra, 2006). The results are summarized in Table 5. Extractionefficiency superior to sonication was observed for both proteins. AmCyanrequired a post extraction high salt wash to release the protein fromthe cell mass; this resulted in a significant purification of theprotein in the salt wash. This is not due to AmCyan retention inextracted cells, since the bulk of the protein was associated with thepellet after either sonication or extraction in the low salt extractionbuffer (i.e. >3 fold improved yield of extraction 2 compared tosonication). The band profile of general E. coli proteins by SDS-PAGEwas similar after extraction or sonication. This demonstrates that mostsoluble E. coli proteins (and soluble recombinant proteins overexpressedin the cell line) are quantitatively extractable from endolysincontaining cells in the low pH extraction buffer, resulting in thebenefits of significant purification (removal of cell mass, gDNA)without cell lysis viscosity issues normally associated with gDNArelease that occurs with homogenization conditions or chemicalextraction. As demonstrated in Example 24, lysozyme can be substitutedfor endolysin in protein extraction.

TABLE 5 Protein extraction Fluores- % Recovery cence (standardizedIntegrity Protein Condition (FU)+ to sonication) (SDS-PAGE) EGFPSonication 3439 100% Single band EGFP Extraction 4660 136% Single bandAmCyan Sonication 3864 100% None detected AmCyan Extraction 99  3% Nonedetected AmCyan Extraction 2† 12,619 337% Single band (purified) †Highsalt wash of pellet from extraction +blanked versus buffer (15fluorescence units = FU).

Example 26 Extraction of Plasmid from Cells Using EDTA, PEI orCombinations

As diagrammed in FIG. 3, extraction of plasmid from cells requirespermeabilization of the outer membrane, inner membrane, and cell wallfor quantitative plasmid extraction. We have demonstrated the utility ofendolysin or lysozyme for permeabilizing the cell wall, and a non ionicdetergent (Triton X-100) for inner membrane solubilization, and havedemonstrated that plasmid extraction efficiency is reduced in theabsence of 1) endolysin or lysozyme, or 2) Triton X-100 (Example 24).Here we investigate the importance of outer membrane permeabilizers inextraction.

A series of 100 g WCW extractions (in 1 L of extraction buffer) usingunfrozen NTC8382-41H-HA plasmid containing fermentation cells wereperformed using a low pH extraction buffer without EDTA (30 mM sodiumacetate, 8% sucrose, pH 5.2) or supplemented with 10 mM EDTA (pH 5.05),50 mM EDTA (pH 4.9) or 50 μg/mL PEI (pH 5.21). After resuspension,sample A was removed, Triton X-100 added to 0.1%, followed by a 10minute incubation at ambient temperature (Sample B), then 20 minutes ina 37° C. bath (Sample C). The entire 1 L extract was centrifuged toremove cells (Sample D). Plasmid was purified from the samplesupernatants using the miniprep kit of Example 24. The results aresummarized in Table 6 and surprisingly demonstrated significant amountsof plasmid can be extracted in the absence of EDTA. The cells had notbeen subjected to a freeze thaw cycle, so the mechanism of outermembrane permeabilization is unknown. Extraction efficiency in low pHextraction buffer without EDTA in cells subjected to a freeze thaw cycleis similar to that in buffers with EDTA.

EDTA chelates Mg⁺⁺ releasing LPS; elimination of EDTA results in lessendotoxin release during extraction (Table 6). An alternative outermembrane permeabilizer, PEI, which does not release endotoxin (LPS)increased extraction efficiency without increasing endotoxin levels inthe final supernatant. This demonstrates alternative outer membranepermeabilization methods can be utilized to release plasmid, and thatsignificant amounts of plasmid are released without any outer membranepermeabilizer.

TABLE 6 EDTA versus PEI extraction efficiency Lysate % plasmid recoveryconcen- (versus Sam- tration 50 mM % gDNA Endotoxin Extraction ple(μg/mL) EDTA) (RT-PCR) Kunits/mL + 50 mM EDTA A 14  5% (positive B 8130% control) C 221 82% D 270 100%  0.26% 2,046 10 mM EDTA A 11  4% B 5922% C 134 50% D 155 57%   0% 4234 0 mM EDTA A 2  1% B 70 26% C 99 37% D100 37% 0.35% 740 0 mM EDTA + A † 2  7% 50 ug/mL PEI B 159 59% C 202 75%D 187 70% 0.46% 467 † Before PEI. After PEI addition 5 μg/mL plasmid +Endotoxin units were determined using the EndoSafe PTS system (CharlesRiver Laboratories, Wilmington MA).

The released plasmid was of a very high purity from all four extractionconditions, with <0.5% of the DNA as gDNA in the supernatant. Asubsequent 0.1% Triton X-100 extraction from frozen cells in 30 mMsodium acetate, 8% sucrose extraction buffer containing 50 μg/mL PEI and1 mM EDTA resulted in 98% extraction yield of intact plasmid with 1.6%gDNA. This demonstrates that extraction is highly selective for pDNAversus gDNA extraction. Preparation of plasmid DNA using the miniprepkit of Example 24 after overnight storage of the 0 mM EDTA extract at 4°C. demonstrated no loss of yield or integrity of plasmid. Thissurprisingly demonstrates that EDTA is not necessary in a low pHextraction buffer to prevent plasmid damage in the extract. This iscontrary to the teachings of Baker and Taylor, Supra, 2003, whereextracted plasmid was demonstrated to be heavily damaged. While notlimiting the application of the embodiments of the invention, this mayindicate that endogenous nucleases cannot function at this pH.Consistent with this, EDTA addition is required to prevent plasmiddegradation upon pH adjustment of the extract to a higher pH (e.g. pH8).

Approximately 1 kg WCW of frozen fermentation cell paste was extractedwith 10 volumes (10 L) 30 mM Sodium Acetate, 8% sucrose extractionbuffer containing 50 μg/mL PEI and 1 mM EDTA resulted in 105% extractionof intact plasmid in the clarified extract, with 1.1% gDNA. The plasmidconcentration in the extract was 638 mg/L. This extraction yield wasdetermined by comparison to analytical alkaline lysis (Qiaprep miniprepspin column kit; Qiagen, Germany). In the analytical assay, plasmid in10 μL of fermentation harvest sample (containing approximately 2 mg WCWcells) are lysed with 250 μL P1, 250 μL P2 and 350 μL N3 (425 volumestotal per volume WCW) and purified and eluted in 2×50 μL elutions as perkit instructions. Lysis at such dilute cell concentration is a highyield analytical method; large scale alkaline lysis yields at morereasonable extraction volumes (40 volumes total, including P1, P2, andP3/N3, per volume WCW) are typically about 50% of this theoreticalyield. This demonstrates, at the multigram scale, that low pH extractionis significantly improved relative to alkaline lysis; yields are muchhigher, in approximately ¼ the total volume, with low gDNA in theextract.

Hoare et al, Supra 2006 report that diatomaceous earth does not bindplasmid at pH 7-10, and can be used as a filter aid in plasmidprocessing in this pH range. The compatibility of the low pH extractionprocess with body feed filtration with diatomaceous earth wasestablished. No plasmid loss after treatment of a 30 mM Sodium acetate,8% sucrose, 50 mM EDTA, 0.1% triton X-100 extract (pH 5.3) with 40 g/Ldiatomaceous earth (Celite 503, JT Baker, Phillipsburg N.J.) wasobserved.

Example 27 Extract Conditioning with Anionic Surfactants and AlkalineEarth Metal Salts

Both the autolysis process and the low pH extraction process releaseplasmid DNA, protein, RNA, LPS, and phospholipids from the cell.Optionally, the extract or lysate can be conditioned to remove one ormore of these components. For example, for protein purification, plasmidDNA could be precipitated by addition of PEI (at higher concentrationsthan is used to permeabilize the cell). Gehart and Daignault, Supra,disclose various methods of conditioning low pH homogenates to removedebris and nucleic acids. These methods could also be applied to the lowpH extraction embodiments of the invention, and are included herein byreference.

For plasmid DNA purification, the released protein, RNA, LPS, andphospholipids components are impurities. We disclose herein a method ofconditioning lysates or extracts to remove one or more of thesecomponents by treatment of the lysate with an anionic surfactant andsequential precipitation with a cationic component (synthetic anionicdetergents). Preferably, the anionic detergent component interacts withlysate or extract components, then the anionic detergent-lysatecomponent complex is precipitated by addition of the cationic component.Preferably the cationic component is an alkaline earth metal. Varioussynthetic anionic detergents and calcium surfactants are reviewed inZapf A, Beck R, Platz G, Hoffmann H. 2003. Advances Colloid andInterface Science 100-102:349-380 and Rodriguez C H, Lowery L H,Scamehorn J F, Harwell J H 2001 Journal Surfactants Detergents 4: 1-14and Rodriguez C H, Chinanasathien C, Scamehron J F, Saiwan C, ChavadejS. 1998 Journal Surfactants Detergents 1: 321-328 and are includedherein by reference. While not limiting the application of the inventionembodiments, a preferred surfactant-alkaline earth metal precipitate isformed by sequential SDS-Calcium treatment to form calcium dodecylsulphate. Other cations than CaCl₂ also form soaps with anionicsurfactants; some are described in Jian-xiao L V, Dong W, Ji-ti Z. J2006 Dispersion Science and Technology 27: 1073-1077 and are includedherein by reference.

We disclose herein application of anionic surfactants to conditionextracts or lysates for plasmid purification by removal of impurities.We disclose that addition of an anionic surfactant such as SDS, followedby addition of a cation such as Ca²⁺, forms insoluble flocculatedcomplexes with the SDS, protein, lipopolysaccharides, and other hostcell impurities (calcium dodecyl sulfate complexes) while leaving thepDNA in solution. The complexes can then be removed by solid-liquidseparation.

The following example demonstrates that formation of surfactant-alkalineearth metal precipitates can be used to remove protein or endotoxin fromplasmid containing cell extracts. A centrifuged extract prepared asdescribed in Example 26 (870 g WCW cell paste and 8.7 L extractionbuffer=30 mM sodium acetate, 8% sucrose, 1 mM EDTA, 50 μg/mL PEI buffer0.1% Triton X-100 pH 5.0, after the extraction, pH 5.46) was conditionedwith SDS-Calcium as follows. SDS was added to 0.2%, and the extractheated to 69° C. and cooled at which time CaCl₂ was added to 14 mM finalconcentration and the extract held overnight. The extract wascentrifuged to remove the calcium dodecyl sulfate complexes. Theconditioned extract contained 106 KEU/mL and 0.27 mg/mL protein (6.6mg/mL protein prior to treatment) with no plasmid loss. SDS-Calciumtreatment of extracts typically reduces endotoxin levels to 50-100KEU/mL. This is a 5-40 fold reduction in endotoxin compared tounconditioned extracts (Table 6). SDS-Calcium treatment will also removeprotein and endotoxin at lower temperatures (i.e. with no heating stepafter SDS addition prior to Calcium addition, or with an optionalcooling step). As well, different concentrations can be used, forexample 0.4% SDS and 28 mM CaCl₂. An investigator skilled in the art candetermine alternative SDS-Calcium treatments wherein SDS-Calcium removesprotein and endotoxin without plasmid loss as well as alternativeanionic surfactant or cationic components.

The application of anionic surfactant-alkaline earth metal treatments tocondition lysates or extracts for plasmid purification has not beentaught in the art. Coffman J L, Shpritzer R I Vicik S M. 2007 WorldPatent Application WO2007035283 disclose flocculation to removecontaminants in protein solutions using combinations of cationic andanionic salts. The application of anionic surfactant-alkaline earthmetal treatments to flocculate or condition cell extracts or lysates isnot contemplated by the inventors. Anionic and cationic salts such asSDS precipitated with ammonium have been utilized to flocculate andremove contaminants from soil DNA isolates (Brolaski M N, Venugopal R J,Stolow D. 2006 World Patent Application WO2006073472) but the inventorsdid not contemplate use of these salts for other applications. SDSprecipitation with high concentrations (1-4 M) ammonium or potassiumsalts is generally utilized in alkaline lysis to precipitate impuritiesand prepare clarified lysates for plasmid processing. In alkaline lysis,SDS-debris complexes are precipitated at final concentrations of 1-4Mammonium or potassium acetate which dramatically increases lysatevolumes and cost. The application of low concentrations of anionicsurfactants and alkaline earth metal salts disclosed herein toflocculate or condition preexisting cell extracts or lysates has notbeen reported.

The following demonstrates that in addition to removal of endotoxin andprotein, SDS-Calcium treatment can unexpectedly be utilized to removeRNA. Approximately 40-50 mL aliquots of a 1 L (100 g WCW cells extractedwith 1 L extraction buffer) centrifuged extract prepared as described inExample 26 (extraction buffer=30 mM sodium acetate, 8% sucrose, 1 mMEDTA, 50 μg/mL PEI buffer 0.1% Triton X-100 pH 5.0, extract pH 5.7) wereconditioned with SDS-Calcium under various pH, temperature and saltconditions. After adjustment of the pH and salt concentration, SDS wasadded to 0.2%, and the extract heated to the target temperature at whichtime CaCl₂ was added to 14 mM final concentration and the extract heatedat temperature a further 10 minutes, followed by cooling to roomtemperature. Aliquots were electrophoresed on a gel, and analyzed forRNA by SYBR Green II prestain and plasmid by SYBR Green I prestain.Plasmid yields were quantified using the kit of Example 24. The resultsare shown in Table 7 and demonstrate novel removal of RNA usingSDS-Calcium and heat treatment. RNA removal with SDS-Calcium heattreatment is robust and does not precipitate plasmid DNA. Aninvestigator skilled in the art can determine alternative buffercompositions wherein SDS-Calcium heat treatment removes RNA withoutplasmid loss. Eon-Duval A, Gumbs K and Ellett C 2003 Biotechnol Bioeng83: 544-553 disclose use of high salt concentration (>0.5 M CaCl₂) toprecipitate RNA from alkaline lysates without plasmid loss. BhikhabhaiR, 1999 European Patent EP0964923 disclose use of high saltconcentration (0.2 M CaCl₂) to precipitate RNA from alkaline lysates.Detraz N J F, Rigaut G. 2006 World Patent Application WO2006060282disclose use of 0.2 M CaCl₂ to precipitation RNA and endotoxin.Collectively, the art teaches that RNA or endotoxin precipitation withCaCl₂ occurs only at high salt concentration (>0.2 M CaCl₂). Thus, whileCaCl₂ precipitation of RNA at high concentrations (0.2 to 1 M) of CaCl₂is taught in the art, RNA removal at low concentrations (e.g. 14 mMCaCl₂) using SDS-Calcium with heating is an unexpected observation thatis not taught in the art.

TABLE 7 SDS-Calcium conditioning of a plasmid extract to remove RNA RNATemperature removal % recovery pH (° C.) NaCl ** plasmid † 5.7 Room tempNot added − 100% (no SDS/Ca) (untreated) 5.5 60 Not added − 65 + 70 +5.7 65 Not Added −  91% 0.05M −  0.1M −  0.2M +/−  0.3M +/−  0.4M +  98% 0.5M +  95%  0.6M +  97% 6.0 60 Not added − 65 +/− 70 + 6.5 60 NotAdded − 65 − 70 + 6.0 60  0.6M + 65 + 70 + † Versus untreated, firstline. ** = no RNA removed, +/− = partial RNA removal, and + = removal ofhigh molecular weight RNA band as revealed by agarose gelelectrophoresis in the presence of Sybrll stain.

Example 28 Improved Solid Liquid Separation by Thermal Flocculation ofan Autolytic Extraction Mixture

Acid Extraction—Thermal Flocculation

Fifty (50) g WCW of autolytic cell paste from a plasmid fermentation(specific plasmid yield=4.6 mg pDNA/g WCW) was mixed with 20 volumes (20L/kg WCW) of Extraction Buffer (30 mM sodium acetate, 10 mM EDTA, 8%sucrose, pH 5.2), and stirred for 20 minutes to ensure completeresuspension. This cell suspension was treated with 0.1% Triton X-100and stirred for 10 more minutes to allow complete release of the plasmidDNA into solution. This extraction mixture was not viscous. A I mLanalytical sample at this point was centrifuged for 5 minutes at 17,000RCF to pellet the solids; analysis of the supernatant at this pointconfirmed complete pDNA release.

The lysate prepared above was passed through a heat exchange apparatusconsisting of a first stainless steel coil immersed in a 70° C. waterbath followed by a second stainless steel coil immersed in an ice waterbath. The flow rate was set such that the residence time, T, in eachcoil was 20 sec. The cooled lysate exiting the heat exchangers wascollected in a bottle. The lysate remained non-viscous, and,surprisingly, much of the cell debris had flocculated and quickly beganto settle. The lysate was stored for about 16 hours at 4° C. FIG. 17 Ashows the resulting lysate, with the settled flocculated cell debris.Approximately 80% of the original volume was recovered after separationof the flocculated material by filtration through two layers ofMiracloth (Calbiochem).

pH 8 Heat Lysis Process

Autolytic cell paste from the same plasmid fermentation was alsoprepared similarly to the continuous heat lysis method described by Zhuet al, Supra, 2005, which describes a lysozyme/heat lysis process at pH8.0. Approximately 50 g WCW of cell paste was resuspended in 20 volumes(about 20 OD₆₀₀) of TE buffer (10 mM Tris, 50 mM EDTA, pH 8.0), andstirred for 10 minutes to completely resuspend the cells. Then the cellsuspension was adjusted to 0.1M NaCl and 2% Triton X-100 (lysozyme wasnot added since this cell paste contained endolysin). After the 20minute incubation, the cell suspension was very viscous. It was giventhe same thermal treatment described in the above paragraph by pumpingthrough the heat exchanger apparatus, and was collected in a bottle.This method did not result in any separation of cell debris byflocculation or sedimentation. This lysate was still slightly viscous.As shown in FIG. 17 B, after 16 hours, the cell debris remainedsuspended throughout the entire volume.

DNA concentration from both lysates was quantified using the kit ofExample 28; the amount of gDNA was quantified by RT-PCR. One (I) mLsamples from before and after the thermal treatments were brieflycentrifuged to obtain clear samples for testing. The results are shownin Table 8.

TABLE 8 Thermal treatment DNA Sample concentration % pDNA % gDNA AcidBefore thermal 0.227 mg/mL 98.8% 1.2% Extraction treatment After thermal0.228 mg/mL 99.0% 1.0% treatment pH 8 heat Before thermal 0.220 mg/mL92.5% 7.5% lysis treatment After thermal 0.244 mg/mL 72.3% 27.7%treatment

This example demonstrates the new and unexpected result of improvedflocculation and sedimentation of cell debris, with greatly reduced gDNAcontamination, achieved by thermal treatment of a crude extract preparedby the low pH extraction procedure.

Agarose gel analysis of 1 μg samples of DNA from both heat treatedlysates is shown in FIG. 18, showing significantly less open circleplasmid and gDNA in the lysate from the low pH extraction process afterheat treatment (lane 1), in comparison to the lysate from the pH 8 heatlysis process (lane 2).

Thus, the reader will see that the endolysins and associated productionprocess embodiments of the invention provide compositions and methodsfor improved plasmid production.

While the above description contains many specifications, these shouldnot be construed as limitations on the invention, but rather as anexemplification of one preferred embodiment thereof. Many othervariations are possible. For example Boyd et al, Supra, 2006 describes aSTET/recombinant lysozyme lysis procedure performed at 20° C. or 37° C.,preferably with an additional alkaline pH shift to denature gDNA. Leeand Sagar, Supra, 2001 describes a heat lysis method that also requiresrecombinant lysozyme. A limitation of both heat and lysozyme lysismethod is the need for large amounts of recombinant lysozyme. Theavailability of recombinant lysozyme, needed for efficient recovery fromboth these processes is an issue for large scale production. Applicationof an endolysin producing cell line embodiment of the invention to thelysozyme lysis process of Boyd et al, Supra, 2006 or heat lysis processof Lee and Sagar, Supra, 2001 would eliminate the need for costlyrecombinant lysozyme.

Accordingly, the scope of the invention should be determined not by theembodiments illustrated, but by the appended claims and their legalequivalents.

We claim:
 1. A fed-batch fermentation method for production ofcovalently closed, super-coiled plasmid DNA, comprising the steps of: a.growing bacterial cells containing a plasmid at a reduced temperatureduring a fed-batch phase, the reduced temperature during the fed-batchphase being approximately 30° C.; b. restricting growth rate of thebacterial cells to approximately μ=0.12 h⁻¹ during the fed-batch phaseby nutrient limitation; c. inducing plasmid production by increasingtemperature to approximately 42° C.; d. continuing growth at elevatedtemperature for up to 15 hours to accumulate plasmid product; and e.holding cells for approximately 0.5 to 3 hrs at a reduced temperatureprior to cell harvest, the reduced temperature prior to cell harvestbeing approximately 25 to 30° C.; whereby said holding cells at areduced temperature of approximately 25-30° C. prior to cell harvestincreases plasmid yield and/or purity above yield and/or purity that areachieved when growing the bacterial cells either without holding cellsat a reduced temperature of approximately 25-30° C. prior to cellharvest or holding cells at a reduced temperature of approximately 15°C. prior to cell harvest.